Methods of treating cancer patients with farnesyltransferase inhibitors

ABSTRACT

The present invention relates to the field of molecular biology and cancer biology. Specifically, the present invention relates to methods of treating a subject with a farnesyltransferase inhibitor (FTI) that include determining whether the subject is likely to be responsive to the FTI treatment based on genotyping and expression profiling of certain immunological genes and RAS mutation status in the subject.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/346,675, filedNov. 8, 2016, which is a continuation of U.S. Ser. No. 15/238,458, filedAug. 16, 2016, which claims the benefit of U.S. Ser. No. 62/206,194filed Aug. 17, 2015, U.S. Ser. No. 62/218,927 filed Sep. 15, 2015, U.S.Ser. No. 62/241,019 filed Oct. 13, 2015, U.S. Ser. No. 62/310,582 filedMar. 18, 2016, and U.S. Ser. No. 62/372,662 filed Aug. 9, 2016, each ofwhich is incorporated herein by reference in its entirety.

FIELD

The present invention relates to the field of molecular biology andcancer biology. Provided herein are methods of using certainimmunologically related genes as biomarkers for predicting clinicalsensitivity and therapeutic response to treatment with afarnesyltransferase inhibitor in a subject having cancer. Furtherprovided herein are kit for carrying out these methods.

BACKGROUND

Stratification of patient populations to improve therapeutic responserate is increasingly valuable in the clinical management of cancerpatients. Farnesyltransferase inhibitors (FTI) are therapeutic agentsthat have utility in the treatment of cancers, such as leukemia,lymphoma and certain solid tumors. However, patients respond differentlyto an FTI treatment. Therefore, methods to predict the responsiveness ofa cancer patient to an FTI treatment, or methods to select patients foran FTI treatment represent unmet needs. The methods and compositions ofthe present invention meet these needs and provide other relatedadvantages.

SUMMARY OF THE INVENTION

Provided herein are methods for population selection of cancer patientsfor treatment with an FTI. The methods provided herein are based, inpart, on the discovery that the genotype and the expression level ofcertain immunological genes can be used to predict responsiveness of acancer patient to an FTI treatment.

In some embodiments, the methods provided herein for treating cancer ina subject include (a) KIR typing the subject, wherein the subject is acarrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeuticallyeffective amount of an FTI to the subject. In some embodiments, thesubject is a carrier of KIR2DS2. In some embodiments, the subject is acarrier of KIR2DS5. In some embodiments, the subject is a carrier ofKIR2DS2 and KIR2DS5.

In some embodiments, the methods provided herein further include HLAtyping the subject before administering the FTI treatment to thesubject, wherein the subject is a carrier of HLA-C2. In some embodiment,the subject is a carrier of both KIR2DS2 and HLA-C2.

In some embodiments, the KIR typing of a subject includes determiningthe presence of a KIR gene in a sample from the subject. In someembodiments, the sample is a blood sample. In some embodiments, thesample is a bone marrow sample. In some embodiments, the sample isperipheral blood mononuclear cells (PBMC). In some embodiments, thesample is enriched natural killer (NK) cells.

In some embodiments, the KIR tying is performed by sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) assay, Immunoblotting assay, orEnzyme-Linked Immunosorbent Assay (ELISA). In one embodiment, the KIRtyping is performed by PCR. In one embodiment, the KIR tying isperformed by DNA microarray. In one embodiment, the KIR typing isperformed by an immunoblotting assay or ELISA.

In some embodiments, the methods provided herein for treating cancer ina subject include (a) determining expression level of a biomarkerselected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5,KIR2DL5, and GZMM in a sample from the subject, wherein (i) theexpression level of KIR2DS2 in the sample is higher than a referenceexpression level of KIR2DS2; (ii) the expression level of KIR2DL2 in thesample is lower than a reference expression level of KIR2DL2; (iii) theexpression level of KIR2DS5 in the sample is higher than a referenceexpression level of KIR2DS5; (iv) the expression level of KIR2DL5 in thesample is lower than a reference expression level of KIR2DL5; or (v) theexpression level of GZMM in the sample is higher than a referenceexpression level of GZMM; or any combination thereof; and (b)administering a therapeutically effective amount of an FTI to thesubject.

In some embodiments, the methods provided herein for treating cancer ina subject include (a) determining expression levels of KIR2DS2 andKIR2DL2, or of KIR2DS5 and KIR2DL5 in a sample from the subject, wherein(i) the ratio of the expression level of KIR2DS2 to the expression levelof KIR2DL2 in the sample is higher than a reference ratio; or (ii) theratio of the expression level of KIR2DS5 to the expression level ofKIR2DL5 in the sample is higher than a reference ratio; and (b)administering a therapeutically effective amount of an FTI to thesubject.

In some embodiments, the sample is a blood sample. In some embodiments,the sample is a bone marrow sample. In some embodiments, the sample isperipheral blood mononuclear cells (PBMC). In some embodiments, thesample is enriched NK cells. In some embodiments, the NK cells arefurther expanded in vitro.

In some embodiments, determining expression level of a biomarkerincludes determining the protein level of the biomarker. Methods ofdetermining the protein level of a biomarker can be animmunohistochemistry (IHC) approach, an immunoblotting assay, flowcytometry (FACS), or ELISA. In some embodiments, the protein level of abiomarker is measured by ELISA.

In some embodiments, determining expression level of a biomarkerincludes determining the mRNA level of the biomarker. Methods ofdetermining the mRNA level of a biomarker can be qPCR, RT-PCR, RNA-seq,microarray analysis, SAGE, MassARRAY technique, or FISH. In someembodiments, the mRNA level of a biomarker is measured by qPCR orRT-PCR.

In some embodiments, the subject is a cancer patient. In someembodiments, the subject has a hematological cancer. In someembodiments, the subject has a solid tumor. The solid tumor can be abenign tumor or a cancer. In some other embodiments, the the subject hasa premalignant condition. The hematological cancer can be acute myeloidleukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocyticleukemia (CMML), natural killer cell lymphoma (NK lymphoma), naturalkiller cell leukemia (NK leukemia), cutaneous T-Cell lymphoma (CTCL),peripheral T-cell lymphoma (PTCL), or chronic myeloid leukemia (CML). Insome embodiments, the patient is a MDS patient. The MDS patient can havevery low risk MDS, low risk MDS, intermediate risk MDS, or high riskMDS. In some embodiments, the patient is a lower risk MDS patient, whichcan have a very low risk MDS, low risk MDS, intermediate risk MDS. Insome embodiments, the patient is an AML patient. In some embodiments,the AML patient is post-remission induction or post transplantation. Insome embodiments, the AML patient is over age 60 or otherwise unfit forremission induction.

In some embodiments, the methods provided herein for selecting a cancerpatient for an FTI treatment include (a) KIR typing the subject, whereinthe subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering atherapeutically effective amount of an FTI to the subject. In someembodiments, the methods a) KIR typing the subject, wherein the subjectis a carrier of KIR2DS2 or KIR2DS5, b) HLA typing the subject, whereinthe subject is a carrier of HLA-C2 and (c) administering atherapeutically effective amount of an FTI to the subject. In someembodiments, the subject is a carrier of both KIR2DS2 and HLA-C2.

In some embodiments, the methods of selecting a cancer patient for anFTI treatment include (a) determining expression level of a biomarkerselected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5,KIR2DL5, and GZMM in a sample from the subject, wherein (i) theexpression level of KIR2DS2 in the sample is higher than a referenceexpression level of KIR2DS2; (ii) the expression level of KIR2DL2 in thesample is lower than a reference expression level of KIR2DL2; (iii) theexpression level of KIR2DS5 in the sample is higher than a referenceexpression level of KIR2DS5; (iv) the expression level of KIR2DL5 in thesample is lower than a reference expression level of KIR2DL5; or (v) theexpression level of GZMM in the sample is higher than a referenceexpression level of GZMM; or any combination thereof; and (b)administering a therapeutically effective amount of an FTI to thesubject.

In some embodiments, the methods provided herein for selecting a cancerpatient for an FTI treatment include (a) determining expression levelsof KIR2DS2 and KIR2DL2, or of KIR2DS5 and KIR2DL5 in a sample from thesubject, wherein (i) the ratio of the expression level of KIR2DS2 to theexpression level of KIR2DL2 in the sample is higher than a referenceratio; or (ii) the ratio of the expression level of KIR2DS5 to theexpression level of KIR2DL5 in the sample is higher than a referenceratio; and (b) administering a therapeutically effective amount of anFTI to the subject.

In one embodiment, the methods provided herein for treating MDS in asubject include (a) KIR typing the subject, wherein the subject is acarrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeuticallyeffective amount of tipifarnib to the subject. The methods can furtherinclude HLA typing the subject, wherein the subject is a carrier ofHLA-C2. In some embodiments, the subject is a carrier of both KIR2DS2and HLA-C2. In some embodiments, the MDS is lower risk MDS.

Provided herein are methods for population selection of cancer patientsfor treatment with an FTI based on Ras mutation status. In someembodiments, provided herein are methods for predicting responsivenessof a cancer patient to an FTI treatment based on the mutation status ofRas in a sample from the patient. In some embodiments, provided hereinare methods for treating cancer in a subject with a therapeuticallyeffective amount of an FTI.

In some embodiments, provided herein are methods for treating a cancerin a subject including (a) determining the presence or absence of a Rasmutation in a sample from the subject, wherein the Ras mutation includesa K-Ras mutation or a N-Ras mutation, and subsequently (b) administeringa therapeutically effective amount of an FTI to the subject if thesample is determined to lack the K-Ras mutation or the N-Ras mutation.

In some embodiments, the methods includes determining the presence orabsence an amino acid substitution at a codon selected from a groupconsisting of G12, G13, and Q61 of K-Ras. In some embodiments, themethods includes determining the presence or absence an amino acidsubstitution at a codon selected from a group consisting of G12, G13,and Q61 of N-Ras.

In some embodiments, the patient is administered an FTI treatment if thesample is determined to not have amino acid substitution at G12, G13,and Q61 of K-Ras, and also not have amino acid substitution at G12, G13,and Q61 of N-Ras. In some embodiments, the patient is administered anFTI treatment if the sample is determined to not have any K-Ras mutationor any N-Ras mutation. In some embodiments, the patient is administeredan FTI treatment if the sample is determined to have wild type K-Ras andwild type N-Ras.

In some embodiments, the subject is a cancer patient. In someembodiments, the subject has a hematological cancer. In someembodiments, the subject has a solid tumor. The solid tumor can be abenign tumor or a cancer. In some other embodiments, the subject has apremalignant condition. The hematological cancer can be chronicmyelomonocytic leukemia (CMML), myeloproliferative neoplasm (MPN),myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), JuvenileMyelomonocytic Leukemia (JMML), chronic myeloid leukemia (CML), naturalkiller cell lymphoma (NK lymphoma), natural killer cell leukemia (NKleukemia), cutaneous T-Cell lymphoma (CTCL), or peripheral T-celllymphoma (PTCL). In some embodiments, the patient is a CMML patient. Insome embodiments, the patient is an MDS patient. In some embodiments,the patient is an AML patient.

In some embodiments, provided herein are methods for treating CMML in asubject (a) determining the presence or absence of a K-Ras mutation in asample from the subject, and subsequently (b) administering atherapeutically effective amount of a tipifarnib to the subject if thesample is determined to have wild type K-Ras.

In some embodiments, provided herein are methods for treating CMML in asubject (a) determining the presence or absence of a N-Ras mutation in asample from the subject, and subsequently (b) administering atherapeutically effective amount of a tipifarnib to the subject if thesample is determined to have wild type N-Ras.

Provided herein are methods for population selection of cancer patientsfor treatment with an FTI based on the presence of a H-Ras mutation. Insome embodiments, provided herein are methods for predictingresponsiveness of a cancer patient to an FTI treatment, methods forcancer patient population selection for an FTI treatment, and methodsfor treating cancer in a subject with a therapeutically effective amountof an FTI, based on the presence of a H-Ras mutation in a sample fromthe patient.

In some embodiments, provided herein are methods for treating a cancerin a subject including (a) determining the presence or absence of aH-Ras mutation in a sample from the subject subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a H-Ras mutation. In someembodiments, the H-Ras mutation can be an amino acid substitution atG12, G13, and Q61 of H-Ras.

In some embodiments, provided herein are methods for treating a cancerin a patient include (a) determining the presence or absence of a H-Rasmutation, a K-Ras mutation, and a N-Ras mutation in a sample from thesubject, and subsequently (b) administering a therapeutically effectiveamount of an FTI to the subject if the sample is determined to have aH-Ras mutation, but no K-Ras mutation or N-Ras mutation. In someembodiments, the methods include (a) determining a cancer patient tohave a H-Ras mutation and wild type K-Ras and wild type N-Ras, andsubsequently (b) administering a therapeutically effective amount of anFTI to the subject. In some embodiments, the FTI is tipifarnib.

In some embodiments, the subject has a hematological cancer. In someembodiments, the subject has a solid tumor. In some embodiments, thecancer is HPV negative. In some embodiments, the cancer is hepatocelluarcarcinoma, head and neck cancer, salivary gland tumor, thyroid tumor,urothelial cancer, breast cancer, melanoma, gastric cancer, pancreaticcancer, or lung cancer. In some embodiments, the cancer is head and necksquamous cell carcinoma (HNSCC). In some embodiments, the cancer issalivary gland tumor. In some embodiments, the cancer is a thyroidtumor.

In some embodiments, provided herein is a method of treating a HNSCC ina subject based on the presence of a H-Ras mutation. In someembodiments, the HNSCC can be HPV negative HNSCC. In some embodiments,the HNSCC can be relapsed/recurrent HNSCC. In some embodiments, theHNSCC can be metastatic HNSCC. The methods provided herein include (a)determining the presence or absence of a H-Ras mutation in a sample fromthe subject having HNSCC, and subsequently (b) administering atherapeutically effective amount of tipifarnib to the subject if thesample is determined to have a H-Ras mutation.

In some embodiments, provided herein is a method of treating a salivarygland tumor in a subject based on the presence of a H-Ras mutation. Insome embodiments, the salivary gland tumor can be HPV negative. In someembodiments, the salivary gland tumor can be advanced salivary glandtumor. In some embodiments, the salivary gland tumor can be metastaticsalivary gland tumor. The methods provided herein include (a)determining the presence or absence of a H-Ras mutation in a sample fromthe subject having salivary gland tumor, and subsequently (b)administering a therapeutically effective amount of tipifarnib to thesubject if the sample is determined to have a H-Ras mutation.

In some embodiments, provided herein is a method of treating a thyroidcancer in a subject based on the presence of a H-Ras mutation. In someembodiments, the thyroid cancer can be HPV negative. In someembodiments, the thyroid cancer can be advanced thyroid cancer. In someembodiments, the thyroid cancer can be metastatic thyroid cancer. Themethods provided herein include (a) determining the presence or absenceof a H-Ras mutation in a sample from the subject having thyroid cancer,and subsequently (b) administering a therapeutically effective amount oftipifarnib to the subject if the sample is determined to have a H-Rasmutation.

In some embodiments, the sample is a tumor biopsy. In some embodiments,the sample is a tissue sample. In some embodiments, the sample is ablood sample. In some embodiments, the sample is a peripheral bloodsample. In some embodiments, the sample is a serum sample. In someembodiments, the sample is a bone marrow sample. In some embodiments,the sample is peripheral blood mononuclear cells (PBMC).

In some embodiments, the Ras mutation status is determined by analyzingnucleic acids obtained from a sample. In some embodiments, the Rasmutation status is determined by analyzing proteins obtained from asample. In some embodiments, the Ras mutation status is determined bysequencing, Polymerase Chain Reaction (PCR), DNA microarray, MassSpectrometry (MS), Single Nucleotide Polymorphism (SNP) assay,denaturing high-performance liquid chromatography (DHPLC), orRestriction Fragment Length Polymorphism (RFLP) assay. In someembodiments, the Ras mutation status is determined by multiplexing PCR.In some embodiments, the Ras mutation status is determined by nextgeneration sequencing.

In some embodiments, the FTI is selected from the group consisting oftipifarnib, lonafarnib (SCH-66336), CP-609,754, BMS-214662, L778123,L744823, L739749, R208176, AZD3409 and FTI-277. In some embodiments, theFTI is administered at a dose of 1-1000 mg/kg body weight. In oneembodiment, the FTI is tipifarnib. In some embodiments, tipifarnib isadministered at a dose of 200-1200 mg twice a day (“b.i.d.”). In someembodiments, tipifarnib is administered at a dose of 600 mg dailyorally. In some embodiments, tipifarnib is administered at a dose of 300mg b.i.d. orally for 3 of out of 4 weeks in repeated 4 week cycles. Insome embodiments, tipifarnib is administered at a dose of 600 mg b.i.d.orally for 3 of out of 4 weeks in repeated 4 week cycles. In someembodiments, tipifarnib is administered at a dose of 900 mg b.i.d.orally in alternate weeks (one week on, one week off) in repeated 4 weekcycles (days 1-7 and 15-21 of repeated 28-day cycles). In someembodiments, tipifarnib is administered at a dose of 1200 mg b.i.d.orally in alternate weeks (days 1-7 and 15-21 of repeated 28-daycycles). In some embodiments, tipifarnib is administered at a dose of1200 mg b.i.d. orally for days 1-5 and 15-19 out of repeated 28-daycycles. In some embodiments, patients receive at least three cycles oftreatment. In some embodiments, patients receive at least six cycles oftreatment.

In some embodiments, the methods provided herein also includeadministering a second therapy to the subject. The second therapy can bea radiation therapy. In some embodiments, the methods provided hereinalso include administering a second therapeutically effective amount ofa secondary active agent or a support care therapy to the subject. Insome embodiments, the secondary active agent is a DNA-hypomethylatingagent, a therapeutic antibody that specifically binds to a cancerantigen, a hematopoietic growth factor, cytokine, anti-cancer agent,antibiotic, cox-2 inhibitor, immunomodulatory agent, anti-thymocyteglobulin, immunosuppressive agent, corticosteroid or a pharmacologicallyderivative thereof. In some embodiments, the secondary active agent is aDNA-hypomethylating agent, such as azacitidine or decitabine. In someembodiments, the second active agent is anti-PD1 antibody or anti-PDL1antibody.

In some embodiments, the kits provided herein for predicting theresponsiveness of a cancer patient to an FTI treatment include at leastone agent for KIR typing the cancer patient, and an ancillary agent,wherein the cancer patient is predicted to be responsive to the FTItreatment if the cancer patient is a carrier of KIR2DS2 or KIR2DS5. Thekits can further include an agent for HLA typing, wherein the cancerpatient is predicted to be responsive to the FTI treatment if the cancerpatient is a carrier of HLA-C2.

In some embodiments, the kits provided herein for predicting theresponsiveness of a cancer patient to an FTI treatment include at leastone agent for determining expression of at least one biomarkers in asample from the cancer patient, and an ancillary agent, wherein thebiomarker is selected from the group consisting of KIR2DS2, KIR2DL2,KIR2DS5, KIR2DL5, and GZMM; and wherein the cancer patient is predictedto be responsive to the FTI treatment if

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5;

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in the sample is higher than a reference ratio; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in the sample is higher than a reference ratio; or anycombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the correlation of KIR2DS5 expression levels with theclinical outcome of AML patients treated with tipifarnib. “SD” standsfor “stable disease”; “PD” stands for “progressive disease”; “CR” standsfor “complete response”; “HI” refers to “hematologic improvement.” FIG.1B shows the correlation of KIR2DS5 expression levels with theprogression-free survival (“PFS”) of AML patients treated withtipifarnib.

FIG. 2A shows the correlation between the ratio of expression level ofKIR2DS2 to the expression level of KIR2DL2 and the PFS of AML patientstreated with tipifarnib. FIG. 2B shows the correlation between the ratioof expression level of KIR2DS2 to the expression level of KIR2DL2 andthe overall survival (“OS”) of AML patients treated with tipifarnib.

FIG. 2A: Cox Proportional Hazards Regression

Parameter b SE Wald P Exp(b) 95% Cl of Exp(b) 2DS/2DL −7.4132 2.001213.7227 0.0002 0.0006 0.0000 to 0.0299

FIG. 2B: Cox Proportional Hazards Regression

b SE Wald P Exp(b) 95% Cl of Exp(b) 2DS2/2DL2 Ratio −5.3430 1.687110.0296 0.0015 0.0048 0.0002 to 0.1283

FIGS. 3A and 3B both show the lack of correlation between the ratio ofexpression level of KIR2DS2 to the expression level of KIR2DL2 and theOS of AML patients treated with non-FTI chemotherapy agents. In FIG. 3A,patients were treated with high dose cytarabine and mitoxantrone. InFIG. 3B, patients were treated with high dose cytarabine andmitoxantrone/intense chemo.

FIG. 4A shows the correlation between the ratio of expression level ofKIR2DS5 to the expression level of KIR2DL5A and both the PFS and OS ofAML patients treated with tipifarnib. FIG. 4B shows the lack ofcorrelation between the ratio of expression level of KIR2DS5 to theexpression level of KIR2DL5A with OS of AML patients treated withnon-FTI chemotherapy agents (cytarabine and mitoxantrone).

FIG. 4A: Cox Proportional Hazards Regression

Tipifarnib/PFS b SE Wald P Exp(b) 95% Cl of Exp(b) 2DS5/2DL5A Ratio−3.5254 1.2351 8.1480 0.0043 0.0294 0.0026 to 0.3272

FIG. 5 shows the correlation of levels of GZMM expression with theclinical outcome of AML patients treated with tipifarnib.

FIG. 6A shows the correlation between the expression level of GZMM andsurvival of AML patients treated with tipifarnib. FIG. 6B shows the lackof correlation between the expression level of GZMM with survival of AMLpatients treated with non-FTI chemotherapy agents (cytarabine andmitoxantrone).

FIG. 6A: Cox Proportional Hazards Regression

(Tipifarnib) b SE Wald P Exp(b) 95% Cl of Exp(b) GZMM/OS −0.5642 0.165211.6675 0.0006 0.5688 0.4122 to 0.7850 GZMM/PFS −0.5856 0.1809 10.47800.0012 0.5568 0.3913 to 0.7923

FIG. 7A shows the correlation of levels of KIR2DS2 expression with theclinical outcome of AML patients treated with tipifarnib. FIG. 7B showsthe specific correlation of levels of KIR2DS2 expression with theclinical outcome of AML patients treated with tipifarnib (left panel),but not with non-FTI chemotherapy agents (right panel).

FIG. 8 shows the correlation between N-RAS wild type status and theprolonged progression-free survival (“PFS”) in AML patients treated withtipifarnib.

FIG. 9 shows the higher response rate to tipifarnib treatment in AMLpatients having wild type N-RAS compared to those having mutant N-RAS.

DETAILED DESCRIPTION 1. DEFINITIONS

As used herein, the articles “a,” “an,” and “the” refer to one or tomore than one of the grammatical object of the article. By way ofexample, a biomarker refers to one biomarker or more than onebiomarkers.

As used herein, the term “NK cell,” or “natural killer cell,” refers tothe type of bone marrow-derived large granular lymphocytes that share acommon progenitor with T cells, but do not have B cell or T cell surfacemarkers. NK cells usually constitute 10-15% of all circulatinglymphocytes. NK cells are defensive cells of innate immunity thatrecognize structures on the surface of virally infected cells or tumorcells and kill these cells by releasing cytotoxins. NK cells can beactivated without previous antigen exposure.

In order to kill infected cells or tumor cells selectively, NK cellsmust distinguish healthy cells from diseased cells. The cytolyticactivity of human NK cells is modulated by the interaction of inhibitoryand activatory membrane receptors, which are expressed on the surface ofNK cells, with MHC (HLA) class I molecules, which are expressed bynon-NK cells, including tumor cells, or cells from a bone marrowtransplant recipient. The killer cell immunoglobulin-like receptors(KIR; or CD158) mapping to chromosome 19q13.4.3-5, constitute a familyof MHC-I (HLA-A, -B, -C) binding receptors that regulate the activationthreshold of NK cells (Valiante el at. Immunity 7:739-751 (1997)).

In humans, the class I HLA complex is about 2000 kb long and containsabout 20 genes. Within the class I region exist genes encoding the wellcharacterized class I MHC molecules designated HLA-A, HLA-B and HLA-C.In addition, there are nonclassical class I genes that include HLA-E,HLA-F, HLA-G, HLA-H, HLA-J and HLA-X as well as a new family known asMIC. While HLA-A and -B play some role, the interactions between KIRsand HLA-C molecules predominate in preventing NK cells from attackinghealthy autologous cells (Colonna et al. PNAS, 90:1200-12004 (1993);Moesta A K et al., Front Immunol. 3:336 (2012)).

HLA-C gene has multiple alleles, including HLA-C1 and HLA-C2 based onthe presence of asparagine or lysine at amino acid position 80 in themature protein (Mandelboim et al. 1996). Furthermore, HLA-C1 contains aconserved serine residue at amino acid position 77, while an asparagineis present in HLA-C2 at the same position. Thus, at least threegenotypes can be distinguished regarding HLA-C: those having both HLA-C1and HLA-C2 (HLA-C1/HLA-C2 heterozygous), those having either HLA-C1(HLA-C1/HLA-C1 homozygous) or HLA-C2 (HLA-C2/HLA-C2 homozygous), andthose lacking both HLA-C1 and HLA-C2.

As used herein, the term “KIR genes” refers to the genes that encode theKIR receptors on NK cells. The KIR genes are clustered in one of themost variable regions of the human genome in terms of both gene contentand sequence polymorphism. This extensive variability generates arepertoire of NK cells in which KIR are expressed at the cell surface ina combinatorial fashion. Interactions between KIR and their appropriateligands on target cells result in the production of positive or negativesignals that regulate NK cell function.

KIR genes are inherited in two major haplotypes: A and B. Haplotype Ahas only one activatory receptor, KIR2DS4, that is inactivated in mostof the US population due to a 22 bp deletion. KIR haplotype B includes22 KIR2DS2 and 16 KIR2DS5 alleles that are present in ˜45% and ˜25% ofCaucasian Americans, respectively. There is a strong linkagedisequilibrium between KIR2DS2 (activatory) and KIR2DL2 (inhibitory).DNA methylation maintains allele-specific KIR gene expression (e.g. CpGisland in KIR2DS2 promoter spans from −160 through +26 and has 6cytosine sites) (Moesta A K et al., Front Immunol. 3:336 (2012)).

To date, at least 14 distinct KIR genes have been identified, which areKIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3,KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1. These genes shareextensive sequence homology. Each gene is about 9-16 Kb in length,divided into 8-9 exons that encode the signal peptide, two or threeextracellular domains, stem, transmembrane region, and cytoplasmic tail.These genes vary with respect to their presence or absence on differentKIR haplotypes, creating considerable diversity in the number of KIRgenotypes observed in the population. For example, some individualsmight carry only seven of the 14 KIR genes while other individuals mightcarry 12 of the 14 KIR genes. Each KIR gene encodes either an inhibitoryor an activating KIR. For example, KIR2DS2 and KIR2DS5 are bothactivating KIRs, and KIR2DL2 and KIR2DL5 are both inhibitory KIRs. Oneparticular KIR gene can have multiple alleles. For example, KIR2DL5includes two alleles, KIR2DL5A and KIR2DL5B. Thus, four genotypes can bedistinguished regarding KIR2DL5: those having both KIR2DL5A andKIR2DL5B, those having either KIR2DL5A or KIR2DL5B, and those lackingKIR2DL5.

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human KIR2DS2 (GENBANK: GQ921920.1; GI: 261362473) areprovided below:

(SEQ ID NO: 1) MSLMVVSMVCVGFFLLQGAWPHEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFEHFLLHREGKYKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDH QEVSYA (SEQ ID NO: 2)ATGTCGCTCATGGTCGTCAGCATGGTGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGCCACATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAGAGGGGAAGTATAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTTTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAACGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCCTATGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCCAAAACCGGTAACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCAAAATCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCTGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTGAACAGCGAGGATTCTGATGAACAAGACCATCAGGAGGTGTCATACGC ATAA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human KIR2DL2 (GENBANK: EU791546.1; GI: 209512828) areprovided below:

(SEQ ID NO: 3) MSLMVVSMACVGFFLLQGAWPHEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFLLHREGKFKDTLHLIGEHEDGVSKANFSIGPMNQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVIGNPSNSWPSPTEPSSKTGNPRHLHILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP (SEQ ID NO: 4)ATGTCGCTCATGGTCGTCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGCCACATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCGCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAGAAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAAGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAATGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCATAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCTAAAACCGGTAACCCCCGACACCTGCACATTCTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCGGTAATGGACCAAGAGTCTGCAGGGAACAGAACAGCGAATAGCGAGGACTCTGATGAACAAGACCCTCAGGAGGTGACATACACACAGTTGAATCACTGCGTTTTCACACAGAGAAAAATCACTCGCCCTTCTCAGAGGCCCAAGACACCCCCAACAGATATCATCGTGTACACGGAACTTCCAAATGCTGAGTCCAGATCCAAAGTTGTCTCCTGCCCATGA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human KIR2DS5 (GENBANK: AJI81015.1; GI: 754367842) areprovided below:

(SEQ ID NO: 5) MSLMVISMACVAFFLLQGAWPHEGFRRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGTFNHTLRLIGEHIDGVSKGNFSIGRMTQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNRTFQADFPLDPATHGGTYRCFGSFRDSPYEWSKSSDPLLVSVTGNSSNSWPSPTEPSSETGNPRHLHVLIGTSVVKLPFTILLFFLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQ EVSYA (SEQ ID NO: 6)ATGTCGCTCATGGTCATCAGCATGGCGTGTGTTGCGTTCTTCTTGCTGCAGGGGGCCTGGCCACATGAGGGATTCCGCAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCATGTTTGAGCACTTCCTTCTGCACAGAGAGGGGACGTTTAACCACACTTTGCGCCTCATTGGAGAGCACATTGATGGGGTCTCCAAGGGCAACTTCTCCATCGGTCGCATGACACAAGACCTGGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCGCCCAGTGACCCTCTGGACATCGTGATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCCTGCAGGGCCCAAGGTCAACAGAACATTCCAGGCCGACTTTCCTCTGGACCCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAAGTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACTCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCCGAAACCGGTAACCCCAGACACCTACACGTTCTGATTGGGACCTCAGTGGTCAAACTCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCATCTGTAATGGACCAAGGGCCTGCGGGGAACAGAACAGTGAACAGGGAGGATTCTGATGAACAGGACCATCAGGAGGTGTCATACGCA TAA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human KIR2DL5A (GENBANK: ABM92655.1 GI: 124245538) areprovided below:

(SEQ ID NO: 7) MSLMVISMACVGFFLLQGAWTHEGGQDKPLLSAWPSAVVPRGGHVTLLCRSRLGFTIFSLYKEDGVPVPELYNKIFWKSILMGPVTPAHAGTYRCRGSHPRSPIEWSAPSNPLVIVVTGLFGKPSLSAQPGPTVRTGENVTLSCSSRSSFDMYHLSREGRAHEPRLPAVPSVDGTFQADFPLGPATHGGTYTCFSSLHDSPYEWSDPSDPLLVSVTGNSSSSSSSPTEPSSKTGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI (SEQ ID NO: 8)ATGTCGCTCATGGTCATCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGACACATGAGGGTGGACAGGACAAGCCCTTGCTGTCTGCCTGGCCCAGCGCTGTGGTGCCTCGAGGAGGACATGTGACTCTTCTGTGTCGCTCTCGTCTTGGGTTTACCATCTTCAGTCTGTACAAAGAAGATGGGGTGCCTGTCCCTGAGCTCTACAACAAAATATTCTGGAAGAGCATCCTCATGGGCCCTGTGACCCCTGCACACGCAGGGACCTACAGATGTCGGGGTTCACACCCGCGCTCCCCCATTGAGTGGTCGGCACCCAGCAACCCCCTGGTGATCGTGGTCACAGGTCTATTTGGGAAACCTTCACTCTCAGCCCAGCCGGGCCCCACGGTTCGCACAGGAGAGAACGTGACCTTGTCCTGCAGCTCCAGGAGCTCATTTGACATGTACCATCTATCCAGGGAGGGGAGGGCCCATGAACCTAGGCTCCCTGCAGTGCCCAGCGTCGATGGAACATTCCAGGCTGACTTTCCTCTGGGCCCTGCCACCCACGGAGGGACCTACACATGCTTCAGCTCTCTCCATGACTCACCCTATGAGTGGTCAGACCCGAGTGACCCACTGCTTGTTTCTGTCACAGGAAACTCTTCAAGTAGTTCATCTTCACCCACTGAACCAAGCTCCAAAACTGGTATCCGCAGACACCTGCACATTCTGATTGGGACCTCAGTGGCTATCATCCTCTTCATCATCCTCTTCTTCTTTCTCCTTCATTGCTGCTGCTCCAACAAAAAGAATGCTGCTGTAATGGACCAAGAGCCTGCCGGGGACAGAACAGTGAACAGGGAGGACTCTGATGATCAAGACCCTCAGGAGGTGACATATGCACAGTTGGATCACTGCGTTTTCACACAGACAAAAATCACTTCCCCTTCTCAGAGGCCCAAGACACCTCCAACAGATACCACCATGTACATGGAACTTCCAAATGCTAAGCCAAGATCATTGTCTCCTGCCCATAAGCACCACAGTCAGGCCTTGAGGGGATCTTCTAGGGAGACAACAGCCCTGTCTCAAAACCGGGTTGCTAGCTCCCA TGTACCAGCAGCTGGAATCTGA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human KIR2DL5B (GENBANK: ABM92657.1 GI: 124245542) areprovided below:

(SEQ ID NO: 9) MSLMVVSMACVGFFLLQGAWTHEGGQDKPLLSAWPSAVVPRGGHVTLLCRSRLGFTIFSLYKEDGVPVPELYNKIFWKSILMGPVTPAHAGTYRCRGSHPRSPIEWSAPSNPLVIVVTGLFGKPSLSAQPGPTVRTGENVTLSCSSRSSFDMYHLSREGRAHEPRLPAVPSVDGTFQADFPLGPATHGGTYTCFSSLHDSPYEWSDPSDPLLVSVTGNSSSSSSSPTEPSSKTGILRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI (SEQ ID NO: 10)ATGTCGCTCATGGTCGTCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGACACATGAGGGTGGACAGGACAAGCCCTTGCTGTCTGCCTGGCCCAGCGCTGTGGTGCCTCGAGGAGGACATGTGACTCTTCTGTGTCGCTCTCGTCTTGGGTTTACCATCTTCAGTCTGTACAAAGAAGATGGGGTGCCTGTCCCTGAGCTCTACAACAAAATATTCTGGAAGAGCATCCTCATGGGCCCTGTGACCCCTGCACACGCAGGGACCTACAGATGTCGGGGTTCACACCCGCGCTCCCCCATTGAGTGGTCGGCACCCAGCAACCCCCTGGTGATCGTGGTCACAGGTCTATTTGGGAAACCTTCACTCTCAGCCCAGCCGGGCCCCACGGTTCGCACAGGAGAGAACGTGACCTTGTCCTGCAGCTCCAGGAGCTCATTTGACATGTACCATCTATCCAGGGAGGGGAGGGCCCATGAACCTAGGCTCCCTGCAGTGCCCAGCGTCGATGGAACATTCCAGGCTGACTTTCCTCTGGGCCCTGCCACCCACGGAGGGACCTACACATGCTTCAGCTCTCTCCATGACTCACCCTATGAGTGGTCAGACCCGAGTGACCCACTGCTTGTTTCTGTCACAGGAAACTCTTCAAGTAGTTCATCTTCACCCACTGAACCAAGCTCCAAAACTGGTATCCTCAGACACCTGCACATTCTGATTGGGACCTCAGTGGCTATCATCCTCTTCATCATCCTCTTCTTCTTTCTCCTTCATTGCTGCTGCTCCAACAAAAAGAATGCTGCTGTAATGGACCAAGAGCCTGCCGGGGACAGAACAGTGAACAGGGAGGACTCTGATGATCAAGACCCTCAGGAGGTGACATATGCACAGTTGGATCACTGCGTTTTCACACAGACAAAAATCACTTCCCCTTCTCAGAGGCCCAAGACACCTCCAACAGATACCACCATGTACATGGAACTTCCAAATGCTAAGCCAAGATCATTGTCTCCTGCCCATAAGCACCACAGTCAGGCCTTGAGGGGATCTTCTAGGGAGACAACAGCCCTGTCTCAAAACCGGGTTGCTAGCTCCC ATGTACCAGCAGCTGGAATCTGA

As used herein, the term “KIR typing” refers to the process ofdetermining the genotype of the KIR genes in a subject, includingdetermining the presence or absence of one or more specific KIR genes oralleles in the genome of the subject. KIR typing can also includedetermining the copy number of one or more specific KIRs genes oralleles in the genome of the subject.

As used herein, the term “HLA typing” refers to the process ofdetermining the genotype of the HLA genes in a subject, includingdetermining the presence or absence of one or more specific HLA genes oralleles in the genome of the subject. HLA typing also includedetermining the copy number of one or more specific HLA genes or allelesin the genome of the subject.

Granzyme M (GZMM) is a serine protease expressed in a multiple cytotoxiclymphocyte subsets. The granule-exocytosis pathway is the majormechanism via which cytotoxic lymphocytes eliminate virus-infected andtumor cells. In this pathway, cytotoxic lymphocytes release granulescontaining the pore-forming protein perforin and a family of serineproteases known as granzymes (GZM) into the immunological synapse.Pore-formation by perforin facilitates entry of granzymes into thetarget cell, where they can activate various death pathways. There arefive human granzymes: GZMA, GZMB, GZMH, GZMK, and GZMM. Of the fiveGZMs, GZMM is a marker for NK cells or NKT cells, and GZMH is a markerfor cytotoxic T cells (Poot, Cell Death and Differentiation 21:359-368(2014)).

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human GZMM (NCBI Ref: NM_020535.3 GI: 65508540) areprovided below:

(SEQ ID NO: 11) MEACVSSLLVLALGALSVGSSFGTQIIGGREVIPHSRPYMASLQRNGSHLCGGVLVHPKWVLTAAHCLAQRMAQLRLVLGLHTLDSPGLTFHIKAAIQHPRYKPVPALENDLALLQLDGKVKPSRTIRPLALPSKRQVVAAGTRCSMAGWGLTHQGGRLSRVLRELDLQVLDTRMCNNSRFWNGSLSPSMVCLAADSKDQAPCKGDSGGPLVCGKGRVLAGVLSFSSRVCTDIFKPPVATAVAPYVS WIRKVTGRSA(SEQ ID NO: 12) ATGGAGGCCTGCGTGTCTTCACTGCTGGTGCTGGCCCTGGGGGCCCTGTCAGTAGGCAGCTCCTTTGGGACCCAGATCATCGGGGGCCGGGAGGTGATCCCCCACTCGCGCCCGTACATGGCCTCACTGCAGAGAAATGGCTCCCACCTGTGCGGGGGTGTCCTGGTGCACCCAAAGTGGGTGCTGACGGCTGCCCACTGCCTGGCCCAGCGGATGGCCCAGCTGAGGCTGGTGCTGGGGCTCCACACCCTGGACAGCCCCGGTCTCACCTTCCACATCAAGGCAGCCATCCAGCACCCTCGCTACAAGCCCGTCCCTGCCCTGGAGAACGACCTCGCGCTGCTTCAGCTGGACGGGAAAGTGAAGCCCAGCCGGACCATCCGGCCGTTGGCCCTGCCCAGTAAGCGCCAGGTGGTGGCAGCAGGGACTCGGTGCAGCATGGCCGGCTGGGGGCTGACCCACCAGGGCGGGCGCCTGTCCCGGGTGCTGCGGGAGCTGGACCTCCAAGTGCTGGACACCCGCATGTGTAACAACAGCCGCTTCTGGAACGGCAGCCTCTCCCCCAGCATGGTCTGCCTGGCGGCCGACTCCAAGGACCAGGCTCCCTGCAAGGGTGACTCGGGCGGGCCCCTGGTGTGTGGCAAAGGCCGGGTGTTGGCCGGAGTCCTGTCCTTCAGCTCCAGGGTCTGCACTGACATCTTCAAGCCTCCCGTGGCCACCGCTGTGGCGCCTTACGTGTCCTGGATCAGGAAGGTCACCGGCCG ATCGGCCTGA

As used herein, the term “subject” refers to a mammal. A subject can bea human or a non-human mammal such as a dog, cat, bovid, equine, mouse,rat, rabbit, or transgenic species thereof. The subject can be apatient, or a cancer patient.

As used herein, the term “cancer” or “cancerous” refers to thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, hematological cancers (e.g., multiple myeloma, lymphoma andleukemia), and solid tumors. As used herein, the term “premalignantcondition” refers to a condition associated with an increased risk ofcancer, which, if left untreated, can lead to cancer. A premalignantcondition can also refer to non-invasive cancer that have not progressedinto aggressive, invasive stage.

As used herein, the term “treat,” “treating,” and “treatment,” when usedin reference to a cancer patient, refer to an action that reduces theseverity of the cancer, or retards or slows the progression of thecancer, including (a) inhibiting the cancer growth, or arrestingdevelopment of the cancer, and (b) causing regression of the cancer, ordelaying or minimizing one or more symptoms associated with the presenceof the cancer.

As used herein, the term “determining” refers to using any form ofmeasurement to assess the presence of a substance, either quantitativelyor qualitatively. Measurement can be relative or absolute. Measuring thepresence of a substance can include determining whether the substance ispresent or absent, or the amount of the substance.

As used herein, the term “carrier” when used in connection with a generefers to a subject whose genome includes at least one copy of the gene,and when used in connection with an allele of a gene refers to a subjectwhose genome includes at least one copy of the specific allele. Forexample, a carrier of KIR2DS2 refers to a subject whose genome includesat least one copy of KIR2DS2. If a gene has more than one alleles, acarrier of the gene refers to subject whose genome includes at least onecopy of at least one allele of the gene. For example, the gene KIR2DL5has two known alleles, KIR2DL5A and KIR2DL5B. A carrier of KIR2DL5Arefers to a subject whose genome includes at least one copy of theallele KIR2DL5A; a carrier of KIR2DL5B refers to a subject whose genomeincludes at least one copy of the allele KIR2DL5B. A carrier of KIR2DL5refers to a subject whose genome includes at least one copy of KIR2DL5A,KIR2DL5B, or both. For another example, a carrier of HLA-C2 refers to asubject whose genome includes at least one copy of the allele HLA-C2.The subject can be HLA-C2/HLA-C2 homozygous, or HLA-C1/HLA-C2heterozygous.

As used herein, the term “administer,” “administering,” or“administration” refers to the act of delivering, or causing to bedelivered, a compound or a pharmaceutical composition to the body of asubject by a method described herein or otherwise known in the art.Administering a compound or a pharmaceutical composition includesprescribing a compound or a pharmaceutical composition to be deliveredinto the body of a patient. Exemplary forms of administration includeoral dosage forms, such as tablets, capsules, syrups, suspensions;injectable dosage forms, such as intravenous (IV), intramuscular (IM),or intraperitoneal (IP); transdermal dosage forms, including creams,jellies, powders, or patches; buccal dosage forms; inhalation powders,sprays, suspensions, and rectal suppositories.

As used herein, the term “therapeutically effective amount” of acompound when used in connection with a disease or disorder refers to anamount sufficient to provide a therapeutic benefit in the treatment ormanagement of the disease or disorder or to delay or minimize one ormore symptoms associated with the disease or disorder. A therapeuticallyeffective amount of a compound means an amount of the compound, alone orin combination with other therapies, that provides a therapeutic benefitin the treatment or management of the disease or disorder. The termencompasses an amount that improves overall therapy, reduces or avoidssymptoms, or enhances the therapeutic efficacy of another therapeuticagent. The term also refers to the amount of a compound thatsufficiently elicits the biological or medical response of a biologicalmolecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system,animal, or human, which is being sought by a researcher, veterinarian,medical doctor, or clinician.

As used herein, the term “sample” refers to a material or mixture ofmaterials containing one or more components of interest. A sample from asubject refers to a sample obtained from the subject, including samplesof biological tissue or fluid origin, obtained, reached, or collected invivo or in situ. A sample can be obtained from a region of a subjectcontaining precancerous or cancer cells or tissues. Such samples can be,but are not limited to, organs, tissues, fractions and cells isolatedfrom a mammal. Exemplary samples include bone marrow, whole blood,partially purified blood, peripheral blood mononuclear cells (“PBMC”),and tissue biopsies. Exemplary samples also include cell lysate, a cellculture, a cell line, a tissue, oral tissue, gastrointestinal tissue, anorgan, an organelle, a biological fluid, a blood sample, a urine sample,a skin sample, and the like.

As used herein, the term “biomarker” refers to a gene that can be eitherpresent or absent in individual subjects, or can be present butdifferentially expressed in individual subjects. The presence abiomarker, including the expression level of the biomarker, in a samplefrom a subject can indicate the responsiveness of the subject to aparticular treatment, such as an FTI treatment.

As used herein, the term “express” or “expression” when used inconnection with a gene refers to the process by which the informationcarried by the gene becomes manifest as the phenotype, includingtranscription of the gene to a messenger RNA (mRNA), the subsequenttranslation of the mRNA molecule to a polypeptide chain and its assemblyinto the ultimate protein.

As used herein, the term “RNA product of the biomarker” refers to a RNAtranscript transcribed from a biomarker, and the term “protein productof the biomarker” refers to a protein or polypeptide translated from aRNA product of a biomarker.

As used herein, the term “expression level” of a biomarker refers to theamount or accumulation of the expression product of a biomarker, suchas, for example, the amount of a RNA product of the biomarker (the RNAlevel of the biomarker) or the amount of a protein product of thebiomarker (the protein level of the biomarker). If the biomarker is agene with more than one alleles, the expression level of a biomarkerrefers to the total amount of accumulation of the expression product ofall existing alleles for this gene, unless otherwise specified. Forexample, the expression level of KIR2DL5 refers to the total expressionlevels of both KIR2DL5A and KIR2DL5B, unless otherwise specified.

As used herein, the term “reference expression level” refers to apredetermined expression level of a biomarker that one can use todetermine the significance of the expression level of the biomarker in asample from a subject. A reference expression level of a biomarker canbe the expression level of the biomarker in a sample from a healthyindividual. A reference expression level of a biomarker can also be acut-off value determined by a person of ordinary skill in the artthrough statistic analysis of the expression levels of the biomarker ina sample population and the responsiveness to a treatment of theindividuals in the sample population. For example, by analyzing theexpression levels of GZMM in individuals of a sample population and theresponsiveness of these individuals to an FTI treatment, a person ofordinary skill in the art can determine a cut-off value as the referenceexpression level of GZMM, wherein a subject is likely to be responsiveto the FTI treatment if the expression level of GZMM of the subject ishigher than the reference expression level.

As used herein, the term “responsiveness” or “responsive” when used inconnection with a treatment refers to the effectiveness of the treatmentin lessening or decreasing the symptoms of the disease being treated.For example, a cancer patient is responsive to an FTI treatment if theFTI treatment effectively inhibits the cancer growth, or arrestsdevelopment of the cancer, causes regression of the cancer, or delays orminimizes one or more symptoms associated with the presence of thecancer in this patient.

The responsiveness to a particular treatment of a cancer patient can becharacterized as a complete or partial response. “Complete response,” or“CR” refers to an absence of clinically detectable disease withnormalization of previously abnormal radiographic studies, bone marrow,and cerebrospinal fluid (CSF) or abnormal monoclonal proteinmeasurements. “Partial response,” or “PR,” refers to at least about a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in allmeasurable tumor burden (i.e., the number of malignant cells present inthe subject, or the measured bulk of tumor masses or the quantity ofabnormal monoclonal protein) in the absence of new lesions.

A person of ordinary skill in the art would understand that clinicalstandards used to define CR, PR, or other level of patientresponsiveness to treatments can vary for different types of cancer. Forexample, for hematopoietic cancers, patient being “responsive” to aparticular treatment can be defined as patients who have a completeresponse (CR), a partial response (PR), or hematological improvement(HI) (Lancet et al., Blood 2:2 (2006)). HI can be defined as any bonemarrow blast count less than 5% or a reduction in bone marrow blasts byat least half. On the other hand, patient being “not responsive” to aparticular treatment can be defined as patients who have eitherprogressive disease (PD) or stable disease (SD). Progressive disease(PD) can be defined as either >50% increase in bone marrow orcirculating blast % from baseline, or new appearance of circulatingblasts (on at least 2 consecutive occasions). Stable disease (SD) can bedefined as any response not meeting CR, PR, HI, or PD criteria.

As used herein, the term “likelihood” refers to the probability of anevent. A subject is “likely” to be responsive to a particular treatmentwhen a condition is met means that the probability of the subject to beresponsive to a particular treatment is higher when the condition is metthan when the condition is not met. The probability to be responsive toa particular treatment can be higher by, for example, 5%, 10%, 25%, 50%,100%, 200%, or more in a subject who meets a particular conditioncompared to a subject who does not meet the condition. For example, acancer patient is “likely” to be responsive to an FTI treatment when thesubject is a carrier of KIR2DS2 means that the probability of a subjectto be responsive to FTI treatment is 5%, 10%, 25%, 50%, 100%, 200%, ormore higher in a subject who is a carrier of KIR2DS2 compared to asubject who is not a carrier of KIR2DS2. For another example, a subjectis “likely” to be responsive to tipifarnib treatment when the expressionlevel of GZMM in a sample from the subject is higher than a referenceexpression level of GZMM means that the probability of a subject to beresponsive to tipifarnib treatment is 5%, 10%, 25%, 50%, 100%, 200%, ormore in a subject whose expression level of GZMM is higher than areference expression level of GZMM compared to a subject whoseexpression level of GZMM is lower than the reference expression level.

Ras proteins are GTPases that regulate proliferation and by transducingbiological information from extracellular signals to the nucleus.Mammalian cells express three ras genes that encode four Ras proteins,which are H-Ras, N-Ras, K_(A)-Ras and K_(B)-Ras. K_(A)-Ras and K_(B)-Rasare also generally referred to as K-Ras. Ras proteins exist in either anactive, GTP-bound or an inactive, GDP-bound, state. Mutant RAS proteinsaccumulate in the GTP-bound conformation due to defective intrinsicGTPase activity and/or resistance to inactivation by GTPase activatingproteins (GAPs). Mutations that lock Ras proteins in their GTP-bound,activated state result in uncontrolled growth and malignanttransformation. K-Ras mutations that result in glycine to valinesubstitutions at the catalytic sites of K-Ras, which leads to the lossof GTPase activity and subsequent continuous binding of GTP to RAS(Yokota, Anti-Cancer Agents in Medicinal Chemistry, 12:163-171 (2012)).The substitution of other amino acids, such as aspartate and valine atcodon 12 and aspartate at codon 13, can result in the projection oflarger amino acid side chains into the GDP/GTP binding pocket of theprotein which interfere with GTP hydrolysis. As a result of thoseconformational and structural changes EGFR signalling becomesderegulated in response to the constitutive activation of K-Ras protein(Herreros-Villanueva et al., Clinica Chimica Acta 431 (2014) 21:1-220).

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human K-Ras Isoform A (K_(A)-Ras) (GENBANK: NM_033360.3GI: 575403058) are provided below:

(SEQ ID NO: 13) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSYRKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLCVFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDLPSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLVREIRQYRLKK ISKEEKTPGC VKIKKCIIM (SEQ ID NO: 14)ATGACTGAAT ATAAACTTGT GGTAGTTGGA GCTGGTGGCGTAGGCAAGAG TGCCTTGACG ATACAGCTAA TTCAGAATCATTTTGTGGAC GAATATGATC CAACAATAGA GGATTCCTACAGGAAGCAAG TAGTAATTGA TGGAGAAACC TGTCTCTTGGATATTCTCGA CACAGCAGGT CAAGAGGAGT ACAGTGCAATGAGGGACCAG TACATGAGGA CTGGGGAGGG CTTTCTTTGTGTATTTGCCA TAAATAATAC TAAATCATTT GAAGATATTCACCATTATAG AGAACAAATT AAAAGAGTTA AGGACTCTGAAGATGTACCT ATGGTCCTAG TAGGAAATAA ATGTGATTTGCCTTCTAGAA CAGTAGACAC AAAACAGGCT CAGGACTTAGCAAGAAGTTA TGGAATTCCT TTTATTGAAA CATCAGCAAAGACAAGACAG AGAGTGGAGG ATGCTTTTTA TACATTGGTGAGGGAGATCC GACAATACAG ATTGAAAAAA ATCAGCAAAGAAGAAAAGAC TCCTGGCTGT GTGAAAATTA AAAAATGCAT TATAATGTAA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human K-Ras Isoform B (K_(B)-Ras) (GENBANK: NM_033360.3GI: 575403058) are provided below:

(SEQ ID NO: 15) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSYRKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLCVFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDLPSRTVDTKQA QDLARSYGIP FIETSAKTRQ GVDDAFYTLVREIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO: 16)ATGACTGAAT ATAAACTTGT GGTAGTTGGA GCTGGTGGCGTAGGCAAGAG TGCCTTGACG ATACAGCTAA TTCAGAATCATTTTGTGGAC GAATATGATC CAACAATAGA GGATTCCTACAGGAAGCAAG TAGTAATTGA TGGAGAAACC TGTCTCTTGGATATTCTCGA CACAGCAGGT CAAGAGGAGT ACAGTGCAATGAGGGACCAG TACATGAGGA CTGGGGAGGG CTTTCTTTGTGTATTTGCCA TAAATAATAC TAAATCATTT GAAGATATTCACCATTATAG AGAACAAATT AAAAGAGTTA AGGACTCTGAAGATGTACCT ATGGTCCTAG TAGGAAATAA ATGTGATTTGCCTTCTAGAA CAGTAGACAC AAAACAGGCT CAGGACTTAGCAAGAAGTTA TGGAATTCCT TTTATTGAAA CATCAGCAAAGACAAGACAG GGTGTTGATG ATGCCTTCTA TACATTAGTTCGAGAAATTC GAAAACATAA AGAAAAGATG AGCAAAGATGGTAAAAAGAA GAAAAAGAAG TCAAAGACAA AGTGTGTAAT TATGTAA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human N-Ras (GENBANK: NM_002524.4 GI: 334688826) areprovided below:

(SEQ ID NO: 17) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSYRKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLCVFAINNSKSF ADINLYREQI KRVKDSDDVP MVLVGNKCDLPTRTVDTKQA HELAKSYGIP FIETSAKTRQ GVEDAFYTLVREIRQYRMKK LNSSDDGTQG CMGLPCVVM (SEQ ID NO: 18)ATGACTGAGT ACAAACTGGT GGTGGTTGGA GCAGGTGGTGTTGGGAAAAG CGCACTGACA ATCCAGCTAA TCCAGAACCACTTTGTAGAT GAATATGATC CCACCATAGA GGATTCTTACAGAAAACAAG TGGTTATAGA TGGTGAAACC TGTTTGTTGGACATACTGGA TACAGCTGGA CAAGAAGAGT ACAGTGCCATGAGAGACCAA TACATGAGGA CAGGCGAAGG CTTCCTCTGTGTATTTGCCA TCAATAATAG CAAGTCATTT GCGGATATTAACCTCTACAG GGAGCAGATT AAGCGAGTAA AAGACTCGGATGATGTACCT ATGGTGCTAG TGGGAAACAA GTGTGATTTGCCAACAAGGA CAGTTGATAC AAAACAAGCC CACGAACTGGCCAAGAGTTA CGGGATTCCA TTCATTGAAA CCTCAGCCAAGACCAGACAG GGTGTTGAAG ATGCTTTTTA CACACTGGTAAGAGAAATAC GCCAGTACCG AATGAAAAAA CTCAACAGCAGTGATGATGG GACTCAGGGT TGTATGGGAT TGCCATGTGT GGTGATGTAA

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human H-Ras (GENBANK: CR536579.1 GI: 49168641) areprovided below:

(SEQ ID NO: 19) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSYRKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLCVFAINNTKSF EDIHQYREQI KRVKDSDDVP MVLVGNKCDLAARTVESRQA QDLARSYGIP YIETSAKTRQ GVEDAFYTLVREIRQHKLRK LNPPDESGPG CMSCKCVLS (SEQ ID NO: 20)ATGACGGAAT ATAAGCTGGT GGTGGTGGGC GCCGGCGGTGTGGGCAAGAG TGCGCTGACC ATCCAGCTGA TCCAGAACCACTTTGTGGAC GAATACGACC CCACTATAGA GGATTCCTACCGGAAGCAGG TGGTCATTGA TGGGGAGACG TGCCTGTTGGACATCCTGGA TACCGCCGGC CAGGAGGAGT ACAGCGCCATGCGGGACCAG TACATGCGCA CCGGGGAGGG CTTCCTGTGTGTGTTTGCCA TCAACAACAC CAAGTCTTTT GAGGACATCCACCAGTACAG GGAGCAGATC AAACGGGTGA AGGACTCGGATGACGTGCCC ATGGTGCTGG TGGGGAACAA GTGTGACCTGGCTGCACGCA CTGTGGAATC TCGGCAGGCT CAGGACCTCGCCCGAAGCTA CGGCATCCCC TACATCGAGA CCTCGGCCAAGACCCGGCAG GGAGTGGAGG ATGCCTTCTA CACGTTGGTGCGTGAGATCC GGCAGCACAA GCTGCGGAAG CTGAACCCTCCTGATGAGAG TGGCCCCGGC TGCATGAGCT GCAAGTGTGT GCTCTCCTGA

Ras isoforms are farnesylated. Farnesyltransferase (FTase) have crucialroles in the post-translational modifications of Ras proteins. A way ofinterfering with Ras function is the inhibition of FTase, the enzymecoupling a 15-carbon isoprenyl group to Ras proteins, byFarnesyltransferase Inhibitors (“FTI”). FTIs are a class of biologicallyactive anticancer drugs that inhibit farnesylation of a wide range oftarget proteins, including Ras. The FTIs block Ras activation throughinhibition of FTase, ultimately resulting in cell growth arrest. Thus,it was predicted that FTIs would be effective therapeutic agents in thetreatment of cancer.

Thirty percent of all human cancers express oncogenically activated Ras.The high prevalence of mutated Ras, found in 30% of all human cancers,makes this pathway an attractive target for anticancer drug development.Initially, it was predicted that the Ras mutation(s) that led toconstitutively active RAS pathway can serve as a biomarker for patientresponse to FTIs, which was based on the preclinical evidence that FTIscould block RAS-transformed cells. (Raponi et al., Blood 111:2589-96(2008)). Contrary to the conventional understanding, disclosed hereinare the unexpected discoveries that the cancer patients who have wildtype K-Ras and N-Ras are more sensitive to FTI treatment compared tothose who have a mutant K-Ras or N-Ras, and that selection of cancerpatients based on the Ras mutation status can improve the overallresponse rate of an FTI treatment, such as a tipifarnib treatment.

As used herein, the term “Ras mutation” refers to an activation mutationin a ras gene or Ras protein. A Ras mutation can refer to either agenetic alternation in the DNA sequence of one of the ras genes thatresults in activation of the corresponding Ras protein, or thealteration in the amino acid sequence of a Ras protein that results inits activation. Thus, the term “Ras mutation” as used herein does notinclude an alternation in a ras gene that does not result in theactivation of the Ras protein, or an alternation of a Ras proteinsequence that does not lead to its activation. Accordingly, a sample ora subject that does not have any “Ras mutation” as used herein can stillhave a mutation in a ras gene that does not affect the activity of theRas protein or a mutation that impairs the activity of the Ras protein,or have a mutation in a Ras protein that does not affect its activity ora mutation that impairs its activity. A sample or a subject can havemultiple copies of a ras gene. A sample or a subject can also have bothwild type and mutant Ras proteins. As used herein, a sample or a subjecthaving a Ras mutation can also have a copy of wild type ras gene and/orthe wild type Ras protein. A sample or a subject that is determined to“have wild type Ras,” as used herein, refers to the sample or subjectthat only has wild type ras gene and the wild type Ras protein, and noRas mutation. Accordingly, a sample or a subject that is determined to“have wild type K-Ras,” as used herein, refers to the sample or subjectthat only has wild type kras gene and wild type K-Ras protein, and noK-Ras mutation. A sample or a subject that is determined to “have wildtype N-Ras,” as used herein, refers to the sample or subject that onlyhas wild type nras gene and wild type N-Ras protein, and no N-Rasmutation.

The Ras protein can be K-Ras, N-Ras, H-Ras, or any combination thereof.The K-Ras can be K_(A)-Ras, K_(B)-Ras, or both. In some embodiments, themutation is a missense mutation that locks the Ras protein into its GTPbound activated state. In some embodiment, the mutation results in anamino acid substitution in one or more of codons 12, 13, 61 of the Rasprotein.

In some embodiments, the Ras mutation is a K-Ras mutation. In someembodiments, the K-Ras mutation is a mutation in K_(A)-Ras, K_(B)-Ras,or both. The K-Ras mutation can include at least one mutation at a codonselected from the group consisting of G12, G13, and Q61 of K_(A)-Ras,K_(B)-Ras, or both. In some embodiments, the K_(A)-Ras mutation caninclude at least one mutation selected from the group consisting of theamino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N,G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V. Insome embodiments, the K_(B)-Ras mutation can include at least onemutation selected from the group consisting of the amino acidsubstitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C,G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the Ras mutation is an N-Ras mutation. In someembodiments, the N-Ras mutation can include at least one mutation at acodon selected from the group consisting of G12, G13, G15, G60 and Q61.In some embodiments, the N-Ras mutation can include at least onemutation at a codon selected from the group consisting of G12, G13, andQ61. In some embodiments, the N-Ras mutation can include at least onemutation selected from the group consisting of the amino acidsubstitutions of G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R,G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the Ras mutation is an H-Ras mutation. In someembodiments, the H-Ras mutation can include at least one mutation at acodon selected from the group consisting of G12, G13, and Q61. In someembodiments, the N-Ras mutation can include at least one mutationselected from the group consisting of the amino acid substitutions ofG12R, G12V, G13C, G13R, Q61L and Q61R.

2. FARNESYLTRANSFERASE INHIBITORS FOR CANCER TREATMENT 2.1.FARNESYLTRANSFERASE INHIBITORS

Provided herein are methods to treat a cancer with an FTI in a selectedcancer patient or a selected population of cancer patients. Therepresentative FTIs roughly belong to two classes (Shen et al., DrugDisc. Today 20:2 (2015)). The FTIs in the first class have the basicframework of farnesyldiphosphate (FPP). For instance, FPP analogs with amalonic acid group (Ta) were reported to be FTIs that compete with FPP(Duez, S. et al. Bioorg. Med. Chem. 18:543-556 (2010)). In addition,imidazole-containing derivatives linked by an acidic substituent and apeptidyl chain were also synthesized as bisubstrate FTIs, and thedesigned bisubstrate inhibitors have better affinities than FPP. TheFTIs in the second class are peptidomimetic molecules, which can bedivided into two groups, namely thiol and non-thiol FTIs. Regarding thethiol FTIs, for instance L-739749, a selective peptidomimetic FTI showspotent antitumor activity in nude mice without system toxicity (Kohl, N.E. et al. PNAS 91:9141-9145 (1994)). Additionally, a variety of thiolinhibitors were also developed, such as tripeptidyl FTIs (Lee, H-Y. etal. Bioorg. Med. Chem. Lett. 12:1599-1602 (2002)).

For non-thiol FTIs, the heterocycles were therefore widely used tosubstitute the thiol group to contact with the zinc ion in the bindingsite. According to the structures of pharmacophoric groups, the nonthiolFTIs can be divided into three classes. The first class is featured bydifferent monocyclic rings, such as L-778123, an FTI in Phase I clinicaltrials for solid tumors and lymphoma. L-778123 binds into the CAAXpeptide site and competes with the CAAX substrate offarnesyltransferase. The second class is represented by tipifarnib inPhase III trials and BMS-214662 in Phase III trials, which are composedof diverse monocyclic rings and bicyclic rings (Harousseau et al. Blood114:1166-1173 (2009)). The representative inhibitor of the third classis lonafarnib, which is active in Ras-dependent and -independentmalignant tumors, and has entered Phase III clinical trials forcombating carcinoma, leukemia, and myelodysplastic syndrome. Lonafarnibis an FTI with a tricycle core, which contains a central seven-memberedring fused with two six-membered aromatic rings.

Thus, FTIs as described herein can take on a multitude of forms butshare the essential inhibitory function of interfering with or lesseningthe farnesylation of proteins implicated in cancer and proliferativediseases.

Numerous FTIs are within the scope of the invention and include thosedescribed in U.S. Pat. Nos. 5,976,851; 5,972,984; 5,972,966; 5,968,965;5,968,952; 6,187,786; 6,169,096; 6,037,350; 6,177,432; 5,965,578;5,965,539; 5,958,939; 5,939,557; 5,936,097; 5,891,889; 5,889,053;5,880,140; 5,872,135; 5,869,682; 5,861,529; 5,859,015; 5,856,439;5,856,326; 5,852,010; 5,843,941; 5,807,852; 5,780,492; 5,773,455;5,767,274; 5,756,528; 5,750,567; 5,721,236; 5,700,806; 5,661,161;5,602,098; 5,585,359; 5,578,629; 5,534,537; 5,532,359; 5,523,430;5,504,212; 5,491,164; 5,420,245; and 5,238,922, the disclosures of whichare hereby incorporated by reference in their entireties.

FTIs within the scope of the invention also include those described inThomas et al., Biologics 1:415-424 (2007); Shen et al., Drug Disc. Today20:2 (2015); Appels et al., The Oncologist 10:565-578 (2005), thedisclosures of which are hereby incorporated by reference in theirentireties.

In some embodiments, the FTIs include Arglabin (i.e.1(R)-10-epoxy-5(S),7(S)-guaia-3(4),11(13)-dien-6,12-olide described inWO-98/28303 (NuOncology Labs); perrilyl alcohol described in WO-99/45912(Wisconsin Genetics); SCH-66336 (lonafarnib), i.e.(+)-(R)-4-[2-[4-(3,10-dibromo-8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide,described in U.S. Pat. No. 5,874,442 (Schering); L778123, i.e.1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,described in WO-00/01691 (Merck); L739749, i.e. compound2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methioninesulfone described in WO-94/10138 (Merck); FTI-277, i.e., methyl{N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate(Calbiochem); L744832, i.e.2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoicacid 1-methylethyl ester (Biomol International L.P.); CP-609,754(Pfizer), i.e.,(R)-6-[(4-chlorophenyl)-hydroxyl-(1-methyl-1-H-imidazol-5-yl)-methyl]-4-(3-ethynylphenyl)-1-methyl-2-(1H)-quinonlinoneand(R)-6-[(4-chlorophenyl)-hydroxyl-(3-methyl-3-H-imidazol-4-yl)-methyl]-4-(3-ethynylphenyl)-1-methyl-2-(1H)-quinolinone;R208176 (Johnson & Johnson), i.e., JNJ-17305457, or(R)-1-(4-chlorophenyl)-1-[5-(3-chlorophenyl)tetrazolo[1,5-a]quinazolin-7-yl]-1-(1-methyl-1H-imidazol-5-yl)methanamine;AZD3409 (AstraZeneca), i.e. (S)-isopropyl2-(2-(4-fluorophenethyl)-5-((((2S,4S)-4-(nicotinoylthio)pyrrolidin-2-yl)methyl)amino)benzamido)-4-(methylthio)butanoate;BMS 214662 (Bristol-Myers Squibb), i.e.(R)-2,3,4,5-tetrahydro-1-(IH-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulphonyl)-1H-1,4-benzodiazapine-7-carbonitrile,described in WO 97/30992 (Bristol Myers Squibb) and Pfizer compounds (A)and (B) described in WO-00/12498 and WO-00/12499.

In some embodiments, the FTI are the non-peptidal, so-called “smallmolecule” therapeutics, such as are quinolines or quinoline derivativesincluding:

7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-2,3-dihydro-o-1H,5H-benzo[ij]quinolizin-5-one,7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-1,2-dihydro-o-4H-pyrrolo[3,2,1-ij]quinoline-4-one,8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl),methyl]-6-(3-chloroph-enyl)-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one, and8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chlorophe-nyl)-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one.

Tipifarnib is a nonpeptidomimetic FTI (Thomas et al., Biologics1:415-424 (2007)). It is a 4,6-disubstituted-1-methylquinolin-2-onederivative((B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-ch-lorophenyl)-1-methyl-2(1H)-quinolinone))that was obtained by optimization of a quinolone lead identified fromcompound library screening. Tipifarnib competitively inhibits the CAAXpeptide binding site of FTase and is extremely potent and highlyselective inhibitor of farnesylation. Tipifarnib is not an inhibition ofgeranylgeranyltransferase I. Tipifarnib has manageable safety profile assingle agent therapy, is reasonably well tolerated in man and requirestwice-daily dosing to obtain effective plasma concentrations.

Tipifarnib is synthesized by the condensation of the anion of1-methylimidazole with a 6-(4-chlorobenzoyl) quinolone derivative,followed by dehydration. The quinolone intermediate was prepared in foursteps by cyclization of N-phenyl-3-(3-chlorophenyl)-2-propenamide,acylation, oxidation and N-methylation. Tipifarnib was identified fromJanssen's ketoconazole and retinoic acid catabolism programs as a keystructural feature into Ras prenylation process. Tipifarnib is a potentinhibitor of FTase in vitro and is orally active in a variety of animalmodels. Single agent activity of tipifarnib was observed in unselectedtumor populations (AML, MDS/CMML, urothelial cancer, breast cancer,PTCL/CTCL) although a phase III clinic study failed to demonstrateimprovement in overall survival.

In some embodiments, provided herein is a method of treating cancer in asubject with an FTI or a pharmaceutical composition having FTI, orselecting a cancer patient for an FTI treatment. The pharmaceuticalcompositions provided herein contain therapeutically effective amountsof an FTI and a pharmaceutically acceptable carrier, diluent orexcipient. In some embodiments, the FTI is tipifarnib; arglabin;perrilyl alcohol; lonafarnib (SCH-66336); L778123; L739749; FTI-277;L744832; R208176; BMS 214662; AZD3409; or CP-609,754. In someembodiments, the FTI is tipifarnib.

2.2. FORMULATIONS

The FTI can be formulated into suitable pharmaceutical preparations suchas solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations or elixirs, for oraladministration or in sterile solutions or suspensions for ophthalmic orparenteral administration, as well as transdermal patch preparation anddry powder inhalers. Typically the FTI is formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition1999).

In the compositions, effective concentrations of the FTI andpharmaceutically acceptable salts is (are) mixed with a suitablepharmaceutical carrier or vehicle. In certain embodiments, theconcentrations of the FTI in the compositions are effective for deliveryof an amount, upon administration, that treats, prevents, or amelioratesone or more of the symptoms and/or progression of cancer, includinghaematological cancers and solid tumors.

The compositions can be formulated for single dosage administration. Toformulate a composition, the weight fraction of the FTI is dissolved,suspended, dispersed or otherwise mixed in a selected vehicle at aneffective concentration such that the treated condition is relieved orameliorated. Pharmaceutical carriers or vehicles suitable foradministration of the FTI provided herein include any such carriersknown to those skilled in the art to be suitable for the particular modeof administration.

In addition, the FTI can be formulated as the sole pharmaceuticallyactive ingredient in the composition or may be combined with otheractive ingredients. Liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as known in the art. Briefly, liposomessuch as multilamellar vesicles (MLV's) may be formed by drying down eggphosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) onthe inside of a flask. A solution of an FTI provided herein in phosphatebuffered saline lacking divalent cations (PBS) is added and the flaskshaken until the lipid film is dispersed. The resulting vesicles arewashed to remove unencapsulated compound, pelleted by centrifugation,and then resuspended in PBS.

The FTI is included in the pharmaceutically acceptable carrier in anamount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described hereinand then extrapolated therefrom for dosages for humans.

The concentration of FTI in the pharmaceutical composition will dependon absorption, tissue distribution, inactivation and excretion rates ofthe FTI, the physicochemical characteristics of the FTI, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art. For example, the amount that is delivered issufficient to ameliorate one or more of the symptoms of cancer,including hematopoietic cancers and solid tumors.

In certain embodiments, a therapeutically effective dosage shouldproduce a serum concentration of active ingredient of from about 0.1ng/ml to about 50-100 μg/ml. In one embodiment, the pharmaceuticalcompositions provide a dosage of from about 0.001 mg to about 2000 mg ofcompound per kilogram of body weight per day. Pharmaceutical dosage unitforms are prepared to provide from about 1 mg to about 1000 mg and incertain embodiments, from about 10 to about 500 mg of the essentialactive ingredient or a combination of essential ingredients per dosageunit form.

The FTI may be administered at once, or may be divided into a number ofsmaller doses to be administered at intervals of time. It is understoodthat the precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro testdata. It is to be noted that concentrations and dosage values may alsovary with the severity of the condition to be alleviated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that the concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions.

Thus, effective concentrations or amounts of one or more of thecompounds described herein or pharmaceutically acceptable salts thereofare mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. Compounds are included in an amount effective forameliorating one or more symptoms of, or for treating, retardingprogression, or preventing. The concentration of active compound in thecomposition will depend on absorption, tissue distribution,inactivation, excretion rates of the active compound, the dosageschedule, amount administered, particular formulation as well as otherfactors known to those of skill in the art.

The compositions are intended to be administered by a suitable route,including but not limited to orally, parenterally, rectally, topicallyand locally. For oral administration, capsules and tablets can beformulated. The compositions are in liquid, semi-liquid or solid formand are formulated in a manner suitable for each route ofadministration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol, dimethylacetamide or other synthetic solvent; antimicrobial agents, such asbenzyl alcohol and methyl parabens; antioxidants, such as ascorbic acidand sodium bisulfate; chelating agents, such asethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Parenteral preparations can be enclosedin ampules, pens, disposable syringes or single or multiple dose vialsmade of glass, plastic or other suitable material.

In instances in which the FTI exhibits insufficient solubility, methodsfor solubilizing compounds can be used. Such methods are known to thoseof skill in this art, and include, but are not limited to, usingcosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such asTWEEN®, or dissolution in aqueous sodium bicarbonate.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablesalts thereof. The pharmaceutically therapeutically active compounds andsalts thereof are formulated and administered in unit dosage forms ormultiple dosage forms. Unit dose forms as used herein refer tophysically discrete units suitable for human and animal subjects andpackaged individually as is known in the art. Each unit dose contains apredetermined quantity of the therapeutically active compound sufficientto produce the desired therapeutic effect, in association with therequired pharmaceutical carrier, vehicle or diluent. Examples of unitdose forms include ampules and syringes and individually packagedtablets or capsules. Unit dose forms may be administered in fractions ormultiples thereof. A multiple dose form is a plurality of identical unitdosage forms packaged in a single container to be administered insegregated unit dose form. Examples of multiple dose forms includevials, bottles of tablets or capsules or bottles of pints or gallons.Hence, multiple dose form is a multiple of unit doses which are notsegregated in packaging.

Sustained-release preparations can also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the compound provided herein,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices includeiontophoresis patches, polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated compound remain in the body for a long time,they may denature or aggregate as a result of exposure to moisture at37° C., resulting in a loss of biological activity and possible changesin their structure. Rational strategies can be devised for stabilizationdepending on the mechanism of action involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non toxic carrier may beprepared. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate or sodium saccharin. Such compositions include solutions,suspensions, tablets, capsules, powders and sustained releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of these compositions are known to those skilled in theart. The contemplated compositions may contain about 0.001% 100% activeingredient, in certain embodiments, about 0.1-85% or about 75-95%.

The FTI or pharmaceutically acceptable salts can be prepared withcarriers that protect the compound against rapid elimination from thebody, such as time release formulations or coatings.

The compositions can include other active compounds to obtain desiredcombinations of properties. The compounds provided herein, orpharmaceutically acceptable salts thereof as described herein, can alsobe administered together with another pharmacological agent known in thegeneral art to be of value in treating one or more of the diseases ormedical conditions referred to hereinabove, such as diseases related tooxidative stress.

Lactose-free compositions provided herein can contain excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose-freecompositions contain an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose-free dosage forms contain an activeingredient, microcrystalline cellulose, pre-gelatinized starch andmagnesium stearate.

Further encompassed are anhydrous pharmaceutical compositions and dosageforms containing a compound provided herein. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are anhydrous ifsubstantial contact with moisture and/or humidity during manufacturing,packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are packaged using materials known to prevent exposure towater such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs and strip packs.

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric coated, sugar coated or film coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms, such ascapsules or tablets. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or compounds of a similarnature: a binder; a diluent; a disintegrating agent; a lubricant; aglidant; a sweetening agent; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, sucrose and starchpaste. Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric coated tablets, because of theenteric coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents may also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are especially useful in the formationof chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted from noneffervescent granules and effervescent preparations reconstituted fromeffervescent granules. Aqueous solutions include, for example, elixirsand syrups. Emulsions are either oil in-water or water in oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used in noneffervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicadd, sodium benzoate and alcohol. Examples of non aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Diluents include lactose and sucrose. Sweetening agentsinclude sucrose, syrups, glycerin and artificial sweetening agents suchas saccharin. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Organic adds include citric and tartaric acid. Sources ofcarbon dioxide include sodium bicarbonate and sodium carbonate. Coloringagents include any of the approved certified water soluble FD and Cdyes, and mixtures thereof. Flavoring agents include natural flavorsextracted from plants such fruits, and synthetic blends of compoundswhich produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is encapsulated ina gelatin capsule. Such solutions, and the preparation and encapsulationthereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and4,410,545. For a liquid dosage form, the solution, e.g., for example, ina polyethylene glycol, may be diluted with a sufficient quantity of apharmaceutically acceptable liquid carrier, e.g., water, to be easilymeasured for administration.

Alternatively, liquid or semi solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include, but are not limited to, those containing acompound provided herein, a dialkylated mono- or poly-alkylene glycol,including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme,tetraglyme, polyethylene glycol-350-dimethyl ether, polyethyleneglycol-550-dimethyl ether, polyethylene glycol-750-dimethyl etherwherein 350, 550 and 750 refer to the approximate average molecularweight of the polyethylene glycol, and one or more antioxidants, such asbutylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propylgallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine,lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoricacid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Parenteral administration, generally characterized by injection, eithersubcutaneously, intramuscularly or intravenously is also providedherein. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol orethanol. In addition, if desired, the pharmaceutical compositions to beadministered may also contain minor amounts of non toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,stabilizers, solubility enhancers, and other such agents, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleateand cyclodextrins. Implantation of a slow release or sustained releasesystem, such that a constant level of dosage is maintained is alsocontemplated herein. Briefly, a compound provided herein is dispersed ina solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The compound diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propyl phydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the FTI is adjusted so that an injection providesan effective amount to produce the desired pharmacological effect. Theexact dose depends on the age, weight and condition of the patient oranimal as is known in the art. The unit dose parenteral preparations arepackaged in an ampule, a vial or a syringe with a needle. Allpreparations for parenteral administration must be sterile, as is knownand practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an FTI is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration.Typically a therapeutically effective dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more,such as more than 1% w/w of the active compound to the treatedtissue(s). The active ingredient may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the tissue being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

The FTI can be suspended in micronized or other suitable form or may bederivatized to produce a more soluble active product or to produce aprodrug. The form of the resulting mixture depends upon a number offactors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They can also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving an FTIprovided herein, or a pharmaceutically acceptable salt thereof, in asuitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. Generally,the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage (including butnot limited to 10-1000 mg or 100-500 mg) or multiple dosages of thecompound. The lyophilized powder can be stored under appropriateconditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, about 1-50 mg, about 5-35 mg, or about 9-30 mg oflyophilized powder, is added per mL of sterile water or other suitablecarrier. The precise amount depends upon the selected compound. Suchamount can be empirically determined.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsion or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The FTI or pharmaceutical composition having an FTI can be formulated asaerosols for topical application, such as by inhalation (see, e.g., U.S.Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosolsfor delivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will have diameters of less than 50 micronsor less than 10 microns.

The FTI or pharmaceutical composition having an FTI can be formulatedfor local or topical application, such as for topical application to theskin and mucous membranes, such as in the eye, in the form of gels,creams, and lotions and for application to the eye or for intracisternalor intraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the active compoundalone or in combination with other pharmaceutically acceptableexcipients can also be administered. These solutions, particularly thoseintended for ophthalmic use, may be formulated as 0.01%-10% isotonicsolutions, pH about 5-7, with appropriate salts.

Other routes of administration, such as transdermal patches, and rectaladministration are also contemplated herein. For example, pharmaceuticaldosage forms for rectal administration are rectal suppositories,capsules and tablets for systemic effect. Rectal suppositories are usedherein mean solid bodies for insertion into the rectum which melt orsoften at body temperature releasing one or more pharmacologically ortherapeutically active ingredients. Pharmaceutically acceptablesubstances utilized in rectal suppositories are bases or vehicles andagents to raise the melting point. Examples of bases include cocoabutter (theobroma oil), glycerin gelatin, carbowax (polyoxyethyleneglycol) and appropriate mixtures of mono, di and triglycerides of fattyacids. Combinations of the various bases may be used. Agents to raisethe melting point of suppositories include spermaceti and wax. Rectalsuppositories may be prepared either by the compressed method or bymolding. An exemplary weight of a rectal suppository is about 2 to 3grams. Tablets and capsules for rectal administration are manufacturedusing the same pharmaceutically acceptable substance and by the samemethods as for formulations for oral administration.

The FTI or pharmaceutical composition having an FTI provided herein canbe administered by controlled release means or by delivery devices thatare well known to those of ordinary skill in the art. Examples include,but are not limited to, those described in U.S. Pat. Nos.: 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595,5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480,5,733,566, 5,739,108, 5,891,474, 5,922,356, 5,972,891, 5,980,945,5,993,855, 6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363,6,264,970, 6,267,981, 6,376,461,6,419,961, 6,589,548, 6,613,358,6,699,500 and 6,740,634, each of which is incorporated herein byreference. Such dosage forms can be used to provide slow orcontrolled-release of FTI using, for example, hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems, multilayer coatings, microparticles, liposomes, microspheres,or a combination thereof to provide the desired release profile invarying proportions. Suitable controlled-release formulations known tothose of ordinary skill in the art, including those described herein,can be readily selected for use with the active ingredients providedherein.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. In one embodiment, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. In certain embodiments,advantages of controlled-release formulations include extended activityof the drug, reduced dosage frequency, and increased patient compliance.In addition, controlled-release formulations can be used to affect thetime of onset of action or other characteristics, such as blood levelsof the drug, and can thus affect the occurrence of side (e.g., adverse)effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic effect over anextended period of time. In order to maintain this constant level ofdrug in the body, the drug must be released from the dosage form at arate that will replace the amount of drug being metabolized and excretedfrom the body. Controlled-release of an active ingredient can bestimulated by various conditions including, but not limited to, pH,temperature, enzymes, water, or other physiological conditions orcompounds.

In certain embodiments, the FTI can be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., thus requiring only afraction of the systemic dose (see, e.g., Goodson, Medical Applicationsof Controlled Release, vol. 2, pp. 115-138 (1984).

In some embodiments, a controlled release device is introduced into asubject in proximity of the site of inappropriate immune activation or atumor. Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990). The F can be dispersed in a solidinner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate,plasticized or unplasticized polyvinylchloride, plasticized nylon,plasticized polyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The active ingredient then diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active ingredientcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the needs of the subject.

The FTI or pharmaceutical composition of FTI can be packaged as articlesof manufacture containing packaging material, a compound orpharmaceutically acceptable salt thereof provided herein, which is usedfor treatment, prevention or amelioration of one or more symptoms orprogression of cancer, including hematological cancers and solid tumors,and a label that indicates that the compound or pharmaceuticallyacceptable salt thereof is used for treatment, prevention oramelioration of one or more symptoms or progression of cancer, includinghematological cancers and solid tumors.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, pens,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment. A wide array offormulations of the compounds and compositions provided herein arecontemplated.

2.3. DOSAGES

In some embodiments, a therapeutically effective amount of thepharmaceutical composition having an FTI is administered orally orparenterally. In some embodiments, the pharmaceutical composition havingtipifarnib as the active ingredient and is administered orally in anamount of from 1 up to 1500 mg/kg daily, either as a single dose orsubdivided into more than one dose, or more particularly in an amount offrom 10 to 1200 mg/kg daily. In some embodiments, the pharmaceuticalcomposition having tipifarnib as the active ingredient and isadministered orally in an amount of 100 mg/kg daily, 200 mg/kg daily,300 mg/kg daily, 400 mg/kg daily, 500 mg/kg daily, 600 mg/kg daily, 700mg/kg daily, 800 mg/kg daily, 900 mg/kg daily, 1000 mg/kg daily, 1100mg/kg daily, or 1200 mg/kg daily. In some embodiments, the FTI istipifarnib.

In some embodiments, the FTI is administered at a dose of 200-1500 mgdaily. In some embodiments, the FTI is administered at a dose of200-1200 mg daily. In some embodiments, the FTI is administered at adose of 200 mg daily. In some embodiments, the FTI is administered at adose of 300 mg daily. In some embodiments, the FTI is administered at adose of 400 mg daily. In some embodiments, the FTI is administered at adose of 500 mg daily. In some embodiments, the FTI is administered at adose of 600 mg daily. In some embodiments, the FTI is administered at adose of 700 mg daily. In some embodiments, the FTI is administered at adose of 800 mg daily. In some embodiments, the FTI is administered at adose of 900 mg daily. In some embodiments, the FTI is administered at adose of 1000 mg daily. In some embodiments, the FTI is administered at adose of 1100 mg daily. In some embodiments, the FTI is administered at adose of 1200 mg daily. In some embodiments, the FTI is administered at adose of 1300 mg daily. In some embodiments, the FTI is administered at adose of 1400 mg daily. In some embodiments, the FTI is tipifarnib.

In some embodiments, the FTI is administered at a dose of 200-1400 mgb.i.d. (i.e., twice a day). In some embodiments, the FTI is administeredat a dose of 300-1200 mg b.i.d. In some embodiments, the FTI isadministered at a dose of 300-900 mg b.i.d. In some embodiments, the FTIis administered at a dose of 600 mg b.i.d. In some embodiments, the FTIis administered at a dose of 700 mg b.i.d. In some embodiments, the FTIis administered at a dose of 800 mg b.i.d. In some embodiments, the FTIis administered at a dose of 900 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1000 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1100 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1200 mg b.i.d. In some embodiments, the FTIis tipifarnib.

As a person of ordinary skill in the art would understand, the dosagevaries depending on the dosage form employed, condition and sensitivityof the patient, the route of administration, and other factors. Theexact dosage will be determined by the practitioner, in light of factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. During a treatmentcycle, the daily dose could be varied. In some embodiments, a startingdosage can be titrated down within a treatment cycle. In someembodiments, a starting dosage can be titrated up within a treatmentcycle. The final dosage can depend on the occurrence of dose limitingtoxicity and other factors. In some embodiments, the FTI is administeredat a starting dose of 300 mg daily and escalated to a maximum dose of400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or1200 mg daily. In some embodiments, the FTI is administered at astarting dose of 400 mg daily and escalated to a maximum dose of 500 mg,600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg daily. Insome embodiments, the FTI is administered at a starting dose of 500 mgdaily and escalated to a maximum dose of 600 mg, 700 mg, 800 mg, 900 mg,1000 mg, 1100 mg, or 1200 mg daily. In some embodiments, the FTI isadministered at a starting dose of 600 mg daily and escalated to amaximum dose of 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mgdaily. In some embodiments, the FTI is administered at a starting doseof 700 mg daily and escalated to a maximum dose of 800 mg, 900 mg, 1000mg, 1100 mg, or 1200 mg daily. In some embodiments, the FTI isadministered at a starting dose of 800 mg daily and escalated to amaximum dose of 900 mg, 1000 mg, 1100 mg, or 1200 mg daily. In someembodiments, the FTI is administered at a starting dose of 900 mg dailyand escalated to a maximum dose of 1000 mg, 1100 mg, or 1200 mg daily.The dose escalation can be done at once, or step wise. For example, astarting dose at 600 mg daily can be escalated to a final dose of 1000mg daily by increasing by 100 mg per day over the course of 4 days, orby increasing by 200 mg per day over the course of 2 days, or byincreasing by 400 mg at once. In some embodiments, the FTI istipifarnib.

In some embodiments, the FTI is administered at a relatively highstarting dose and titrated down to a lower dose depending on the patientresponse and other factors. In some embodiments, the FTI is administeredat a starting dose of 1200 mg daily and reduced to a final dose of 1100mg, 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, or 300 mgdaily. In some embodiments, the FTI is administered at a starting doseof 1100 mg daily and reduced to a final dose of 1000 mg, 900 mg, 800 mg,700 mg, 600 mg, 500 mg, 400 mg, or 300 mg daily. In some embodiments,the FTI is administered at a starting dose of 1000 mg daily and reducedto a final dose of 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, or300 mg daily. In some embodiments, the FTI is administered at a startingdose of 900 mg daily and reduced to a final dose of 800 mg, 700 mg, 600mg, 500 mg, 400 mg, or 300 mg daily. In some embodiments, the FTI isadministered at a starting dose of 800 mg daily and reduced to a finaldose of 700 mg, 600 mg, 500 mg, 400 mg, or 300 mg daily. In someembodiments, the FTI is administered at a starting dose of 600 mg dailyand reduced to a final dose of 500 mg, 400 mg, or 300 mg daily. The dosereduction can be done at once, or step wise. In some embodiments, theFTI is tipifarnib. For example, a starting dose at 900 mg daily can bereduced to a final dose of 600 mg daily by decreasing by 100 mg per dayover the course of 3 days, or by decreasing by 300 mg at once.

A treatment cycle can have different length. In some embodiments, atreatment cycle can be one week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, or 12 months. In someembodiments, a treatment cycle is 4 weeks. A treatment cycle can haveintermittent schedule. In some embodiments, a 2-week treatment cycle canhave 5-day dosing followed by 9-day rest. In some embodiments, a 2-weektreatment cycle can have 6-day dosing followed by 8-day rest. In someembodiments, a 2-week treatment cycle can have 7-day dosing followed by7-day rest. In some embodiments, a 2-week treatment cycle can have 8-daydosing followed by 6-day rest. In some embodiments, a 2-week treatmentcycle can have 9-day dosing followed by 5-day rest.

In some embodiments, the FTI is administered daily for 3 of out of 4weeks in repeated 4 week cycles. In some embodiments, the FTI isadministered daily in alternate weeks (one week on, one week off) inrepeated 4 week cycles. In some embodiments, the FTI is administered ata dose of 300 mg b.i.d. orally for 3 of out of 4 weeks in repeated 4week cycles. In some embodiments, the FTI is administered at a dose of600 mg b.i.d. orally for 3 of out of 4 weeks in repeated 4 week cycles.In some embodiments, the FTI is administered at a dose of 900 mg b.i.d.orally in alternate weeks (one week on, one week off) in repeated 4 weekcycles. In some embodiments, the FTI is administered at a dose of 1200mg b.i.d. orally in alternate weeks (days 1-7 and 15-21 of repeated28-day cycles). In some embodiments, the FTI is administered at a doseof 1200 mg b.i.d. orally for days 1-5 and 15-19 out of repeated 28-daycycles.

In some embodiments, a 900 mg b.i.d. tipifarnib alternate week regimencan be used adopted. Under the regimen, patients receive a starting doseof 900 mg, po, b.i.d. on days 1-7 and 15-21 of 28-day treatment cycles.In some embodiments, patients receive two treatment cycles. In someembodiments, patients receive three treatment cycles. In someembodiments, patients receive four treatment cycles. In someembodiments, patients receive five treatment cycles. In someembodiments, patients receive six treatment cycles. In some embodiments,patients receive seven treatment cycles. In some embodiments, patientsreceive eight treatment cycles. In some embodiments, patients receivenine treatment cycles. In some embodiments, patients receive tentreatment cycles. In some embodiments, patients receive eleven treatmentcycles. In some embodiments, patients receive twelve treatment cycles.In some embodiments, patients receive more than twelve treatment cycles.

In the absence of unmanageable toxicities, subjects can continue toreceive the tipifarnib treatment for up to 12 months. The dose can alsobe increased to 1200 mg b.i.d. if the subject is tolerating thetreatment well. Stepwise 300 mg dose reductions to controltreatment-related, treatment-emergent toxicities can also be included.

In some other embodiments, tipifarnib is given orally at a dose of 300mg b.i.d. daily for 21 days, followed by 1 week of rest, in 28-daytreatment cycles (21-day schedule; Cheng D T, et al., J Mol Diagn.(2015) 17(3):251-64). In some embodiments, a 5-day dosing ranging from25 to 1300 mg b.i.d. followed by 9-day rest is adopted (5-day schedule;Zujewski J., J Clin Oncol., (2000) February; 18(4):927-41). In someembodiments, a 7-day b.i.d. dosing followed by 7-day rest is adopted(7-day schedule; Lara P N Jr., Anticancer Drugs., (2005) 16(3):317-21;Kirschbaum M H, Leukemia., (2011) October; 25(10):1543-7). In the 7-dayschedule, the patients can receive a starting dose of 300 mg b.i.d. with300 mg dose escalations to a maximum planned dose of 1800 mg b.i.d. Inthe 7-day schedule study, patients can also receive tipifarnib b.i.d. ondays 1-7 and days 15-21 of 28-day cycles at doses up to 1600 mg b.i.d.

FTI can inhibit the growth of mammalian tumors when administered as atwice daily dosing schedule. Administration of an FTI in a single dosedaily for one to five days can produce a marked suppression of tumorgrowth lasting out to at least 21 days. In some embodiments, FTI isadministered at a dosage range of 50-400 mg/kg. In some embodiments, FTIis administered at 200 mg/kg. Dosing regimen for specific FTIs are alsowell known in the art (e.g., U.S. Pat. No. 6,838,467, which isincorporated herein by reference in its entirety). For example, suitabledosages for the compounds Arglabin (WO98/28303), perrilyl alcohol (WO99/45712), SCH-66336 (U.S. Pat. No. 5,874,442), L778123 (WO 00/01691),2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methioninesulfone (WO94/10138), BMS 214662 (WO 97/30992), AZD3409; Pfizercompounds A and B (WO 00/12499 and WO 00/12498) are given in theaforementioned patent specifications which are incorporated herein byreference or are known to or can be readily determined by a personskilled in the art.

In relation to perrilyl alcohol, the medicament may be administered 1-4g per day per 150 lb human patient. Preferably, 1-2 g per day per 150 lbhuman patient. SCH-66336 typically can be administered in a unit dose ofabout 0.1 mg to 100 mg, more preferably from about 1 mg to 300 mgaccording to the particular application. Compounds L778123 and1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinonemay be administered to a human patient in an amount between about 0.1mg/kg of body weight to about 20 mg/kg of body weight per day,preferably between 0.5 mg/kg of bodyweight to about 10 mg/kg of bodyweight per day.

Pfizer compounds A and B may be administered in dosages ranging fromabout 1.0 mg up to about 500 mg per day, preferably from about 1 toabout 100 mg per day in single or divided (i.e. multiple) doses.Therapeutic compounds will ordinarily be administered in daily dosagesranging from about 0.01 to about 10 mg per kg body weight per day, insingle or divided doses. BMS 214662 may be administered in a dosagerange of about 0.05 to 200 mg/kg/day, preferably less than 100 mg/kg/dayin a single dose or in 2 to 4 divided doses.

2.4. COMBINATION THERAPIES

In some embodiments, the FTI treatment is administered in combinationwith radiotherapy, or radiation therapy. Radiotherapy includes usingγ-rays, X-rays, and/or the directed delivery of radioisotopes to tumorcells. Other forms of DNA damaging factors are also contemplated, suchas microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and4,870,287; all of which are hereby incorporated by references in theirentireties), and UV-irradiation. It is most likely that all of thesefactors affect a broad range of damage on DNA, on the precursors of DNA,on the replication and repair of DNA, and on the assembly andmaintenance of chromosomes.

In some embodiments, a therapeutically effective amount of thepharmaceutical composition having an FTI is administered thateffectively sensitizes a tumor in a host to irradiation. (U.S. Pat. No.6,545,020, which is hereby incorporated by reference in its entirety).Irradiation can be ionizing radiation and in particular gamma radiation.In some embodiments, the gamma radiation is emitted by linearaccelerators or by radionuclides. The irradiation of the tumor byradionuclides can be external or internal.

Irradiation can also be X-ray radiation. Dosage ranges for X-rays rangefrom daily doses of 50 to 200 roentgens for prolonged periods of time (3to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

In some embodiments, the administration of the pharmaceuticalcomposition commences up to one month, in particular up to 10 days or aweek, before the irradiation of the tumor. Additionally, irradiation ofthe tumor is fractionated the administration of the pharmaceuticalcomposition is maintained in the interval between the first and the lastirradiation session.

The amount of FTI, the dose of irradiation and the intermittence of theirradiation doses will depend on a series of parameters such as the typeof tumor, its location, the patients' reaction to chemo- or radiotherapyand ultimately is for the physician and radiologists to determine ineach individual case.

In some embodiments, the methods provided herein further includeadministering a therapeutically effective amount of a second activeagent or a support care therapy. The second active agent can be achemotherapeutic agent. A chemotherapeutic agent or drug can becategorized by its mode of activity within a cell, for example, whetherand at what stage they affect the cell cycle. Alternatively, an agentcan be characterized based on its ability to directly cross-link DNA, tointercalate into DNA, or to induce chromosomal and mitotic aberrationsby affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine,navelbine, transplatinum, and pharmaceutically acceptable salts, acids,or derivatives of any of the above.

The second active agents can be large molecules (e.g., proteins) orsmall molecules (e.g., synthetic inorganic, organometallic, or organicmolecules). In some embodiments, the second active agent is aDNA-hypomethylating agent, a therapeutic antibody that specificallybinds to a cancer antigen, a hematopoietic growth factor, cytokine,anti-cancer agent, antibiotic, cox-2 inhibitor, immunomodulatory agent,anti-thymocyte globulin, immunosuppressive agent, corticosteroid or apharmacologically active mutant or derivative thereof.

In some embodiments, the second active agent is a DNA hypomethylatingagent, such as a cytidine analog (e.g., azacitidine) or a5-azadeoxycytidine (e.g. decitabine). In some embodiments, the secondactive agent is a cytoreductive agent, including but not limited toInduction, Topotecan, Hydrea, PO Etoposide, Lenalidomide, LDAC, andThioguanine. In some embodiments, the second active agent isMitoxantrone, Etoposide, Cytarabine, or Valspodar. In some embodiment,the second active agent is Mitoxantrone plus Valspodar, Etoposide plusValspodar, or Cytarabine plus Valspodar. In some embodiment, the secondactive agent is idarubicin, fludarabine, topotecan, or ara-C. In someother embodiments, the second active agent is idarubicin plus ara-C,fludarabine plus ara-C, mitoxantrone plus ara-C, or topotecan plusara-C. In some embodiments, the second active agent is a quinine. Othercombinations of the agents specified above can be used, and the dosagescan be determined by the physician.

For any specific cancer type described herein, treatments as describedherein or otherwise available in the art can be used in combination withthe FTI treatment. For example, drugs that can be used in combinationwith the FTI include belinostat (Beleodaq®) and pralatrexate (Folotyn®),marketed by Spectrum Pharmaceuticals, romidepsin (Istodax®), marketed byCelgene, and brentuximab vedotin (Adcetris®) (for ALCL), marketed bySeattle Genetics; drugs that can be used in combination with the FTIinclude azacytidine (Vidaza®) and lenalidomide (Revlimid®), marketed byCelgene, and decitabine (Dacogen®) marketed by Otsuka and Johnson &Johnson; drugs that can be used in combination with the FTI for thyroidcancer include AstraZeneca's vandetanib (Caprelsa®), Bayer's sorafenib(Nexavar®), Exelixis' cabozantinib (Cometriq®) and Eisai's lenvatinib(Lenvima®).

Non-cytotoxic therapies such as tpralatrexate (Folotyn®), romidepsin(Istodax®) and belinostat (Beleodaq®) can also be used in combinationwith the FTI treatment.

In some embodiments, the second active agent is an immunotherapy agent.In some embodiments, the second active agent is anti-PD1 antibody oranti-PDL1 antibody.

In some embodiments, it is contemplated that the second active agent orsecond therapy used in combination with an FTI can be administeredbefore, at the same time, or after the FTI treatment. In someembodiments, the second active agent or second therapy used incombination with an FTI can be administered before the FTI treatment. Insome embodiments, the second active agent or second therapy used incombination with an FTI can be administered at the same time as FTItreatment. In some embodiments, the second active agent or secondtherapy used in combination with an FTI can be administered after theFTI treatment.

The FTI treatment can also be administered in combination with a bonemarrow transplant. In some embodiments, the FTI is administered beforethe bone marrow transplant. In other embodiments, the FTI isadministered after the bone marrow transplant.

3. IMMUNOLOGICAL GENES AS BIOMARKERS FOR FTI TREATMENT

Provided herein are methods of selection of cancer patients fortreatment with a farnesyltransferase inhibitor (FTI). The methodsprovided herein are based, in part, on the discovery that the genotypesand the expression levels of certain genes that are associated withactivities of natural killer cells (NK cells) are correlated with theclinical benefit of an FTI treatment. Specifically, the genotyping ofKIR genes and HLA genes and the expression levels of biomarkersincluding KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM can be used topredict the responsiveness of a cancer patient to an FTI treatment. Asprovided herein, in addition to the expression levels of the individualbiomarkers, the relative ratio of expression levels between certainbiomarkers, for example, the ratio of expression level of KIR2DS2 tothat of KIR2DL2, or the ratio of expression level of KIR2DS5 to that ofKIR2DL5, can also be used to predict responsiveness of a cancer patientto an FTI treatment. Accordingly, provided herein are methods forpredicting responsiveness of a cancer patient to an FTI treatment,methods for cancer patient population selection for an FTI treatment,and methods for treating cancer in a subject with a therapeuticallyeffective amount of an FTI, based on the genotype or the expressionlevels of these biomarkers in a sample from the patient. Also providedherein are compositions and kits for predicting responsiveness of acancer patient to an FTI treatment.

Farnesyltransferase (FTase) have crucial roles in the post-translationalmodifications of Ras proteins. FTIs are a class of biologically activeanticancer drugs that inhibit farnesylation of a wide range of targetproteins, including Ras. The Ras proteins play a pivotal role in thetransduction of cell growth-stimulating signals, and mutation of the rasgene leads to constant activation of the protein, ultimately resultingin uncontrolled cell proliferation. The high prevalence of mutated rasgenes, found in 30% of all human cancers, makes this pathway anattractive target for anticancer drug development. A way of interferingwith Ras function is the inhibition of FTase, the enzyme coupling a15-carbon isoprenyl group to Ras proteins, by FTIs. The FTIs block Rasactivation through inhibition of FTase, ultimately resulting in cellgrowth arrest. Thus, it was predicted that FTIs would be effectivetherapeutic agents in the treatment of cancer.

However, no correlation between ras mutations and response to FTIs wasdemonstrated in past clinical studies (Karp et al. Blood 97:3361-3369(2001); and US. Patent Pub. 20070048782)). While several early clinicalstudies focused on cancers that exhibited high frequencies of rasmutations, the response rate was disappointingly low in those trials.(Mesa Lancet Oncol 6:279-286 (2006); Rao et al. J Clin Oncol22:3950-3957 (2004))

Early studies of tipifarnib, an FTI, were conducted in poor risk andpreviously untreated AML patients (CTEP-20 phase II), and AML patientswith relapsed/refractory AML (INT-17 Phase II). A phase III study oftipifarnib versus best supportive care (BSC) failed to demonstrateimprovement in overall survival. Multiple gene/proteins have beenassociated in the literature with the activity of FTI (AKAP13, mDIA,etc.) (Raponi et al. Clin Cancer Res. 13:2254-60 (2007); Kamasani et al.Cancer Biology & Therapy, 6:1418-1423 (2007)), and analyses of geneexpression profiling in bone marrow samples from 2 AML studies (CTEP-20,INT-17) identified the ratio of the expression of 2 genes: RASGRP1 (Tcell signal transducer) and APTX (DNA repair protein) as a potentialbiomarker of tipifarnib's activity in AML (Raponi et al. Blood.111:2589-96 (2008)). However, a subsequent prospective study using the2-gene ratio in bone marrow blasts as inclusion criterion failed todemonstrate significant clinical benefit of tipifarnib in AML (Lancet etal. Blood (ASH) 120: Abstract 1508 (2012)).

The present invention identifies multiple immunological genes asbiomarkers associated with better prognosis for an FTI treatment, andnovel methods are provided herein for patient selection for an FTItreatment. Unlike previously identified markers, such as RASGRP1, whichwas found to be associated with good prognosis in not only FTItreatment, but also other standard chemotherapy, the immunologicalrelated biomarkers identified in instant invention are specificallyassociated with clinic benefit of an FTI treatment, but not agents ofother standard chemotherapies.

The biomarkers as identified herein include KIR2DS2, KIR2DL2, KIR2DS5,KIR2DL5, GZMM, as well as the specific ligand for KIR2DS2, HLA-C2.Carriers of KIR2DS2 and HLA-C2 were shown to be predisposed to MDS.(Serio et al., Blood (ASH Annual Meeting Abstracts) 2006 108: Abstract2670; Cook et al., Blood 2004; 103: 1521-152) The immunologicalbiomarkers identified in the instant invention are all NK cell relatedgenes. The discovery that an FTI, such as tipifarnib, selectivelytargets cancers with the specific KIR genotypes or expression profilesdescribed herein can be, at least in part, based on the mechanism thatcertain NK cells with the specific KIR genotypes or expression profilescan induce autoimmunity. Such NK cells can also down regulate antigenpresentation, kill or down regulate certain subtypes of T cells. Throughinhibiting the KIR-RAS signaling, an FTI can modulate or inhibit theactivity of NK cells, facilitate the cytotoxity toward cancer cells bymodulating the patient's own immune system. Also, through inhibiting theKIR-RAS signaling, an FTI can modulate or inhibit the activity of NKcells and other immune cells against normal hematological cells andtheir precursors, reducing or eliminating the need for red blood cell orplatelet transfusion, or hematological growth factor administration.

3.1. KIR TYPING AND HLA TYPING

As provided herein, the genotype of KIR genes and HLA genes of a subjectcan be indicative of the likelihood of the subject to respond to an FTItreatment. A cancer patient who is a carrier of KIR2DS2, KIR2DS5, orHLA-C2 is likely to be responsive to an FTI treatment. Accordingly, KIRtyping cancer patients, and selectively treating those who are carriersof KIR2DS2 or KIR2DS5, can increase the overall response rate of thecancer patients to an FTI treatment. In addition, HLA typing the cancerpatients, and selectively treating those who are carrier of HLA-C2, canfurther increase the overall response rate of the cancer patients to anFTI treatment.

In some embodiments, provided herein are methods for treating cancer ina subject by administering a therapeutically effective amount of an FTIto the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5.In some embodiments, provided herein is a method for treating cancer ina subject by KIR typing the subject, and administering a therapeuticallyeffective amount of an FTI to the subject, wherein the subject is acarrier of KIR2DS2 or KIR2DS5. In some embodiments, the subject is acarrier of KIR2DS2. In some embodiments, the subject is a carrier ofKIR2DS5. In some embodiments, the subject is a carrier of both KIR2DS2and KIR2DS5. In some embodiments, the subject is also not a carrier ofKIR2DL2. In some embodiments, the subject is also not a carrier ofKIR2DL5. In some embodiments, the subject is a carrier of KIR2DS2, butnot a carrier of KIR2DL2. In some other embodiments, the subject is acarrier of KIR2DS5, but not a carrier of KIR2DL5.

In some embodiments, the methods for treating cancer in a subject asprovided herein further include HLA typing the subject, andadministering a therapeutically effective amount of an FTI to thesubject who is a carrier of HLA-C2. In some embodiments, the subject isa carrier of both KIR2DS2 and HLA-C2. In some embodiments, the subjectis a carrier of both KIR2DS5 and HLA-C2. In some embodiments, thesubject is a carrier of KIR2DS2, KIR2DS5 and HLA-C2. In someembodiments, the subject who is a carrier of HLA-C2 is HLA-C2/HLA-C2homozygous. In some embodiments, the subject is HLA-C1/HLA-C2heterozygous.

In some embodiments, provided herein is a method for selecting a cancerpatient for an FTI treatment by KIR typing, wherein a cancer patient isselected for the FTI treatment if the cancer patient is a carrier ofKIR2DS2 or KIR2DS5. In some embodiments, provided herein is a method forpredicting the likelihood of a cancer patient to be responsive to an FTItreatment by KIR typing, and determining that the cancer patient islikely to be responsive to an FTI treatment if the cancer patient is acarrier of KIR2DS2 or KIR2DS5. In some embodiments, the method furtherincludes administering a therapeutically effective amount of an FTI tothe cancer patient. In some embodiments, the subject is a carrier ofKIR2DS2. In some embodiments, the subject is a carrier of KIR2DS5. Insome embodiments, the subject is a carrier of both KIR2DS2 and KIR2DS5.In some embodiments, the subject is also not a carrier of KIR2DL2. Insome embodiments, the subject is also not a carrier of KIR2DL5. In someembodiments, the subject is a carrier of KIR2DS2, but not a carrier ofKIR2DL2. In some other embodiments, the subject is a carrier of KIR2DS5,but not a carrier of KIR2DL5.

In some embodiments, the methods for selecting a cancer patient for anFTI treatment or predicting the likelihood of a cancer patient to beresponsive to an FTI treatment as provided herein further include HLAtyping the subject, and administering a therapeutically effective amountof an FTI to the subject who is a carrier of HLA-C2. In someembodiments, the subject is a carrier of both KIR2DS2 and HLA-C2. Insome embodiments, the subject is a carrier of both KIR2DS5 and HLA-C2.In some embodiments, the subject is a carrier of KIR2DS2, KIR2DS5 andHLA-C2. In some embodiments, the subject who is a carrier of HLA-C2 isHLA-C2/HLA-C2 homozygous. In some embodiments, the subject isHLA-C1/HLA-C2 heterozygous.

Methods of KIR typing are well known in the art. Exemplary methods ofKIR typing are disclosed in WO 2012047985; Lebedeva et al., Hum Immun.,68(9):789-96 (2007); Gonzalez et al., Hum Immun., 70(10):858-63 (2009);Yun et al., Blood (ASH Annual Meeting Abstracts) 106:Abstract 1407(2005) (Also see Yun et al., Clin Immunol. 123(3):272-280 (2007).);Leung et al., J Immun. 174:6540-6545 (2005); Dinauer et al., US2008/0280289 (See also WO 2005/046459 selected parts; and KIR GenotypingProduct Brochure 2004.); Chen et al., WO 2009/051672. Also seePCT/US2008/011671; Trachtenberg et al, Patent Application PublicationNo. US 2008/0213787 (Also see WO 2007/041067.); Houtchens et al.,Immunogenetics. 59(7):525-37 (2007).; Gomez-Lozano et al., TissueAntigens 59(3):184-193 (2002); and Shilling et al., J Immunol.168:2307-2315 (2002); U.S. Pat. Nos. 6,723,564, 6,111,251, 6,104,028,6,558,902, 6,706,530, 6,423,966, 5,777,324, 6,569,385, 6,500,621,6,300,076, and 6,258,538; Uhrberg et al., Immunity 7:753-763 (1997);Gomez-Lozano et al., Tissue Antigens 59:184-193 (2002); Cook et al.,Hum. Immunology 64:567-571 (2003); Crum et al., Tissue Antigens56:313-326 (2000); Middleton et al., Transplant immunology 10:147-164(2002); Ross et al., Nature Biotech., 16:1347-1351 (1998); Fei et al.,Rapid Comm. Mass. Spec., 14:950-959 (2000); Fei et al., NAR26(11):2827-2828 (1998); Amexis et al., PNAS 98(21) 12097-12102 (2001);Li et al., Electrophoresis 20:1258-1265 (1999); Buetow et al., PNAS98(2) 581-584 (2001); Storm et al., Methods in Mol. Biol., 212:241-262(2003); Parham, Immunology Lett. 92:11-13 (2004); and MassARRAY™Homogenous Mass EXTEND™ (hME) Assay, Sequenom®, Application Notes,Bulletin #1021; each of which are hereby incorporated by reference intheir entirety.

Moreover, some KIR genotyping kits available include, Inno-Train,“KIR-Ready Gene” Product Brochure September 2005; Miltenyi Biotec, “KIRTyping Kit” Product Brochure 2009; Invitrogen, “KIR Genotyping SSP Kit”Product Brochure November 2006; and Tepnel Lifecodes, “KIR Genotyping”Product Brochure June 2005, each of which are hereby incorporated byreference in their entirety.

The methods provided herein can be performed by any method describedherein or otherwise known in the art. In some embodiments, providedherein is a method for treating cancer in a subject with an FTI by KIRtyping, or selecting a cancer patient for an FTI treatment by KIRtyping, wherein the KIR typing is performed by sequencing, PolymeraseChain Reaction (PCR), DNA microarray, Mass Spectrometry (MS), SingleNucleotide Polymorphism (SNP) assay, Immunoblotting assay, orEnzyme-Linked Immunosorbent Assay (ELISA). In some embodiments, the KIRtyping is performed by DNA microarray. In some embodiments, the KIRtyping is performed by ELISA. In some embodiments, the KIR typing isperformed by sequencing. In some embodiments, the KIR typing isperformed by next generation sequencing (NGS).

In some embodiments, the KIR typing can be performed by PCR. In someembodiments, KIR typing can be performed by PCR using sequence specificprimer (SSP). In some embodiments, the SSPs include those that arespecific for amplifying KIR2DL2, KIR2DL5, KIR2DS2, KIR2DS5, or anycombination thereof. In some embodiments, KIR typing can be performed byPCR using sequence-specific oligonucleotide probe (SSOP). In someembodiments, KIR typing can be performed by PCR using sequence basedtyping (SBT). In some embodiments, KIR typing can be performed by DNAmicroarray. In some embodiments, KIR typing can be performed by MS. Insome embodiments, the KIR typing can be performed by matrix-assistedlaser desorption/ionization time-of-flight (MALDI-TOF) massspectrometry. As a person of ordinary skill in the art would understand,the KIR typing can be performed by any method described herein orotherwise known in the art.

Methods for HLA typing are well known in the art. Initially, the mostextensively employed DNA typing method for the identification of thesealleles has been restriction fragment length polymorphism (RFLP)analysis. In addition to restriction fragment length polymorphism(PCR-RFLP), another approach has been the hybridization of PCR amplifiedproducts with sequence-specific oligonucleotide probes (PCR-SSO) todistinguish between HLA alleles (see, Tiercy et al., (1990) Blood Review4: 9-15, which is hereby incorporated by reference in its entirety).This method requires a PCR product of the HLA locus of interest beproduced and then dotted onto nitrocellulose membranes or strips. Theneach membrane is hybridized with a sequence specific probe, washed, andthen analyzed by exposure to x-ray film or by colorimetric assaydepending on the method of detection. Similar to the PCR-SSPmethodology, probes are made to the allelic polymorphic area responsiblefor the different HLA alleles. Each sample must be hybridized and probedat least 100-200 different times for a complete Class I and II typing.Hybridization and detection methods for PCR-SSO typing include the useof non-radioactive labeled probes, microplate formats, etc. (see e.g.,Saiki et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86: 6230-6234; Erlichet al. (1991) Eur. J. Immunogenet. 18(1-2): 33-55; Kawasaki et al.(1993) Methods Enzymol. 218:369-381; all of which are herebyincorporated by reference in their entireties).

A molecular typing method using sequence specific primer amplification(PCR-SSP) has been described (see, Olerup and Zetterquist (1992) TissueAntigens 39: 225-235). This PCR-SSP method is simple, useful and fast,since the detection step is much simpler. In PCR-SSP, allelic sequencespecific primers amplify only the complementary template allele,allowing genetic variability to be detected with a high degree ofresolution. This method allows determination of HLA type simply bywhether or not amplification products (collectively called an“amplicon”) are present or absent following PCR. In PCR-SSP, detectionof the amplification products is usually done by agarose gelelectrophoresis followed by ethidium bromide (EtBr) staining of the gel.

Another HLA typing method is SSCP—Single-Stranded ConformationalPolymorphism. Briefly, single stranded PCR products of the different HLAloci are run on non-denaturing Polyacrylamide Gel Electrophoresis(PAGE). The single strands will migrate to a unique location based ontheir base pair composition. By comparison with known standards, atyping can be deduced. It can be used to determine true homozygosity.

Other methods of HLA typing, including but not limited to sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) Assay, Immunoblotting assay, orEnzyme-Linked Immunosorbent Assay (ELISA), can also be used in themethods provided herein. In some embodiments, the sequencing can be NGS.Other methods have been described in U.S. Pat. No. 6,670,124, U.S. Pat.No. 5,468,611, U.S. Pat. No. 8,435,740, U.S. Pat. No. 8,771,951 and U.S.20130267613; which are hereby incorporated by reference in theirentireties. Other different methods for HLA typing known in the art canalso be used in methods provided herein.

For example, Single Nucleotide Polymorphism (SNP) Assay can be used forHLA typing. The SNP assay can type different HLA based on polymorphismat position 77 in HLA-C and position 83 in HLA-B and -A. The SNP assaycan be performed on the HT7900 from Applied Biosystems, following theallelic discrimination assay protocol provided by the manufacturer.Primers for the assay were designed in such a way that they amplifiedall the alleles of a particular HLA type (such as HLA-C) as well as theamplicon containing the polymorphic region of interest. Two probes weredesigned with a single mismatch between them. Each probe bound only onegroup of alleles and was labeled with either 6FAM or VIC fluorescent dyeat their 5′ end. The probes also contained Taqman® minor groove binder(MGB) with non-fluorescent quencher (NFQ) (Applied Biosystems). ForHLA-C, forward primer can be 5′-TTGGGACCGGGAGACACAG-3′ (SEQ ID NO: 46)and reverse primer can be 5′-CGATGTAATCCTTGCCGTC-3′ (SEQ ID NO: 47). Theprobes used for HLA-C1 and HLA-C2 can be 6FAM-CCGAGTGAGCCTGC-MGBNFQ (SEQID NO: 48) and VIC-CCGAGTGAACCTGC-MGBNFQ (SEQ ID NO: 49), respectively.Each assay reaction mix can contain 250 nM probe concentration and 20 ngof genomic DNA in 1× Taqman genotyping master mix from AppliedBiosystems (USA).

In some embodiments, the KIR typing or HLA typing is performed as acompanion diagnostic to the FTI treatment. The companion diagnostic canbe performed at the clinic site where the subject is treated. Thecompanion diagnostic can also be performed at a site separate from theclinic site where the subject is treated.

In some embodiments, the methods of KIR typing or HLA typing includeobtaining a sample from a subject. The subject can be a cancer patient.The sample can be a whole blood sample, a bone marrow sample, apartially purified blood sample, PBMCs, or tissue biopsy. In someembodiments, the sample is a bone marrow sample from a cancer patient.In some embodiments, the sample is PBMCs from a cancer patient. In someembodiments, the sample is enriched NK cells. The NK cells can beenriched from bone marrow, whole blood, or partially purified blood froma cancer patient. In some embodiments, the NK cells are further expandedin vitro before KIR typing.

3.2. KIR EXPRESSION AND GZMM EXPRESSION

As provided herein, the expression level of a biomarker selected fromthe group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM ina sample from a subject can be indicative of the likelihood of thesubject to be responsive to an FTI treatment. A cancer patient whoseexpression level of KIR2DS2 is higher than a reference expression levelof KIR2DS2 is likely to be responsive to an FTI treatment. A cancerpatient whose expression level of KIR2DS5 is higher than a referenceexpression level of KIR2DS5 is likely to be responsive to an FTItreatment. A cancer patient whose expression level of GZMM is higherthan a reference expression level of GZMM is likely to be responsive toan FTI treatment. A cancer patient whose expression level of KIR2DL2 islower than a reference expression level of KIR2DL2 is likely to beresponsive to an FTI treatment. A cancer patient whose expression levelof KIR2DL5 is lower than a reference expression level of KIR2DL5 islikely to be responsive to an FTI treatment. Accordingly, detecting theexpression level of one or more of these biomarkers in cancer patients,and selectively treating the cancer patients who meet one or more of theabove-described conditions, can increase the overall response rate ofthe cancer patients to an FTI treatment.

In some embodiments, the expression level of KIR2DL5 is the totalexpression levels of KIR2DL5A and KIR2DL5B. In some embodiments, theexpression level of KIR2DL5 is the expression level of KIR2DL5A. In someembodiments, the expression level of KIR2DL5 is the expression level ofKIR2DL5B.

Additionally, provided herein are methods of using the ratio ofexpression levels of certain biomarkers to predict the likelihood of asubject to be responsive to an FTI treatment. For example, a high ratioof expression level of KIR2DS2 to the expression level of KIR2DL2 (the“2DS2/2DL2 ratio”) can indicate that the subject is likely to beresponsive to an FTI treatment. Similarly, a high ratio of expressionlevel of KIR2DS5 to the expression level of KIR2DL5 (the “2DS5/2DL5ratio”) can indicate that the subject is likely to be responsive to anFTI treatment. Accordingly, detecting the expression level of thesebiomarkers in cancer patients, and selectively treating the cancerpatients whose 2DS2/2DL2 ratio is higher than a reference ratio, orwhose 2DS5/2DL5 ratio is higher than a reference ratio, or both, canincrease the overall response rate of cancer patients to the FTItreatment.

In some embodiments, provided herein is a method for treating cancer ina subject by administering a therapeutically effective amount of an FTIto the subject, wherein

(i) the expression level of KIR2DS2 in a sample from the subject ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the subject islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the subject ishigher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the subject islower than a reference expression level of KIR2DL5; or

(v) the expression level of GZMM in a sample from the subject is higherthan a reference expression level of GZMM; or any combination thereof.

In some embodiments, provided herein is a method for treating cancer ina subject by

-   (a) determining expression level of a biomarker selected from the    group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in    a sample from the subject, wherein

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5; or

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM; and

-   (b) administering a therapeutically effective amount of an FTI to    the subject.

In some embodiments, provided herein is a method for selecting a cancerpatient for an FTI treatment by determining the expression level of abiomarker selected from the group consisting of KIR2DS2, KIR2DL2,KIR2DS5, KIR2DL5, and GZMM in a sample from a cancer patient, whereinthe cancer patient is selected for the FTI treatment if

(i) the expression level of KIR2DS2 in a sample from the subject ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the subject islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the subject ishigher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the subject islower than a reference expression level of KIR2DL5; or

(v) the expression level of GZMM in a sample from the subject is higherthan a reference expression level of GZMM; or any combination thereof.

In some embodiments, methods provided herein include treating a subjectwith a therapeutically effective amount of an FTI or selecting a cancerpatient for an FTI treatment, wherein one of the five conditions is met:

(i) the expression level of KIR2DS2 in the sample of the subject orcancer patient is higher than a reference expression level of KIR2DS2(condition 1);

(ii) the expression level of KIR2DS5 in the sample of the subject orcancer patient is higher than a reference expression level of KIR2DS5(condition 2);

(iii) the expression level of KIR2DL2 in the sample of the subject orcancer patient is lower than a reference expression level of KIR2DL2(condition 3);

(iv) the expression level of KIR2DL5 in the sample of the subject orcancer patient lower than a reference expression level of KIR2DL5(condition 4); and

(v) the expression level of GZMM in the sample of the subject or cancerpatient is higher than a reference expression level of GZMM (condition5).

A person of ordinary skill in the art would understand, satisfaction ofany one of the five above described conditions can indicate that asubject is likely to be responsive to an FTI treatment. Accordingly,each condition can independently serve as a patient selection criterionfor an FTI treatment in order to increase the overall response rate. Aperson of ordinary skill in the art would also understand thatcombinations of two or more conditions can also serve as patientselection criteria for FTI treatment, which are more selective than asingle condition and can potentially achieve higher overall responserate. Accordingly, also provided herein are method of using anycombination or permutation of the above conditions for patient selectionfor an FTI treatment.

In some embodiments, the method provided herein includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the subject or cancerpatient meets two of the five above described conditions, such asconditions 1 and 2, 1 and 3, 1 and 4, 1 and 5, 2 and 3, 2 and 4, 2 and5, 3 and 4, 3 and 5, or 4 and 5. In some embodiments, the subject orcancer patient meets conditions 1 and 2. In some embodiments, thesubject or cancer patient meets conditions 1 and 3. In some embodiments,the subject or cancer patient meets conditions 1 and 4. In someembodiments, the subject or cancer patient meets conditions 1 and 5. Insome embodiments, the subject or cancer patient meets conditions 2 and3. In some embodiments, the subject or cancer patient meets conditions 2and 4. In some embodiments, the subject or cancer patient meetsconditions 2 and 5. In some embodiments, the subject or cancer patientmeets conditions 3 and 4. In some embodiments, the subject or cancerpatient meets conditions 3 and 5. In some embodiments, the subject orcancer patient meets conditions 4 and 5. For example, in someembodiments, the method includes treating a subject with atherapeutically effective amount of an FTI or selecting a cancer patientfor an FTI treatment, wherein expression level of KIR2DS2 in a sample ofthe subject is higher than a reference expression level of KIR2DS2, andwherein the expression level of GZMM in a sample of the subject ishigher than a reference expression level of GZMM. For another example,in some embodiments, the method includes treating a subject with atherapeutically effective amount of an FTI or selecting a cancer patientfor an FTI treatment, wherein the expression level of KIR2DS5 in asample of the subject is higher than a reference expression level ofKIR2DS5, and the expression level of KIR2DL2 in a sample of the subjectis lower than a reference expression level of KIR2DL2.

In some embodiments, the method provided herein includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the subject or cancerpatient meets three of the five above described conditions, such asconditions 1, 2 and 3; 1, 2 and 4; 1, 2 and 5; 1, 3 and 4; 1, 3 and 5;1, 4 and 5; 2, 3 and 4; 2, 3 and 5; 2, 4 and 5; or 3, 4 and 5. In someembodiments, the subject or cancer patient meets conditions 1, 2 and 3.In some embodiments, the subject or cancer patient meets conditions 1, 2and 4. In some embodiments, the subject or cancer patient meetsconditions 1, 2 and 5. In some embodiments, the subject or cancerpatient meets conditions 1, 3 and 4. In some embodiments, the subject orcancer patient meets conditions 1, 3 and 5. In some embodiments, thesubject or cancer patient meets conditions 1, 4 and 5. In someembodiments, the subject or cancer patient meets conditions 2, 3 and 4.In some embodiments, the subject or cancer patient meets conditions 2, 3and 5. In some embodiments, the subject or cancer patient meetsconditions 2, 4 and 5. In some embodiments, the subject or cancerpatient meets conditions 3, 4 and 5. For an example, in some otherembodiments, the method includes treating a subject with atherapeutically effective amount of an FTI or selecting a cancer patientfor an FTI treatment, wherein the expression level of KIR2DS2 in asample of the subject is higher than a reference expression level ofKIR2DS2, the expression level of KIR2DL2 in a sample of the subject islower than a reference expression level of KIR2DL2, and the expressionlevel of GZMM in a sample of the subject is higher than a referenceexpression level of GZMM.

In some embodiments, the method provided herein includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the subject or cancerpatient meets four of the five above described conditions, such asconditions 1, 2, 3 and 4; 1, 2, 3 and 5; 1, 2, 4 and 5; 1, 3, 4 and 5;or 2, 3, 4 and 5. In some embodiments, the subject or cancer patientmeets conditions 1, 2, 3 and 4. In some embodiments, the subject orcancer patient meets conditions 1, 2, 3 and 5. In some embodiments, thesubject or cancer patient meets conditions 1, 2, 4 and 5. In someembodiments, the subject or cancer patient meets conditions 1, 3, 4 and5. In some embodiments, the subject or cancer patient meets conditions2, 3, 4 and 5.

In some embodiments, the method provided herein includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the subject or cancerpatient meets all five above described conditions, namely, wherein

(i) the expression level of KIR2DS2 in a sample from the subject ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the subject islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the subject ishigher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the subject islower than a reference expression level of KIR2DL5; and

(v) the expression level of GZMM in a sample from the subject is higherthan a reference expression level of GZMM.

In addition to the expression level of individual biomarkers, the ratioof expression levels between two biomarkers can also serve as criterionfor patient selection for an FTI treatment to increase response rate. Insome embodiments, provided herein is a method for treating cancer in asubject by administering a therapeutically effective amount of an FTI tothe subject, wherein

(i) the ratio of the expression level of KIR2DS2 to the expression levelof KIR2DL2 (the 2DS2/2DL2 ratio) in a sample from the subject is higherthan a reference 2DS2/2DL2 ratio; or

(ii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 (the 2DS5/2DL5 ratio) in a sample from the sample ishigher than a reference 2DS5/2DL5 ratio.

In some embodiments, provided herein is a method of treating cancer in asubject by determining expression levels of KIR2DS2 and KIR2DL2, orexpression levels of KIR2DS5 and KIR2DL5 in a sample from the subject,wherein

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; and administering a therapeutically effective amount ofan FTI to the subject.

In some embodiments, provided herein is a method for selecting a cancerpatient for an FTI treatment by determining expression levels of KIR2DS2and KIR2DL2, or expression levels of KIR2DS5 and KIR2DL5 in a samplefrom a cancer patient, wherein the cancer patient is selected for theFTI treatment if

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; and administering a therapeutically effective amount ofan FTI to the subject.

In some embodiments, the method provided herein includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the 2DS2/2DL2 ratio in asample from the subject or cancer patient is higher than a reference2DS2/2DL2 ratio. In some embodiments, the method of present inventionincludes treating a subject with a therapeutically effective amount ofan FTI or selecting a cancer patient for an FTI treatment, wherein the2DS5/2DL5 ratio in a sample from the subject or cancer patient is higherthan a reference 2DS5/2DL5 ratio. In some embodiments, the method ofpresent invention includes treating a subject with a therapeuticallyeffective amount of an FTI or selecting a cancer patient for an FTItreatment, wherein the 2DS2/2DL2 ratio in a sample from the subject orcancer patient is higher than a reference 2DS2/2DL2 ratio, and 2DS5/2DL5ratio in a sample from the subject or cancer patient is higher than areference 2DS5/2DL5 ratio.

In some embodiments, the methods provided herein for selecting a cancerpatient for an FTI treatment can also be based on both the expressionlevel of individual biomarkers as well as the ratio of expression levelbetween biomarkers. In some embodiments, the method includes treating asubject with a therapeutically effective amount of an FTI or selecting acancer patient for an FTI treatment, wherein the 2DS2/2DL2 ratio in asample from the subject or cancer patient is higher than a reference2DS2/2DL2 ratio, and the subject or cancer patient meets at least one ofthe five above described conditions regarding individual expressionlevel of the biomarkers. In some embodiments, the method includestreating a subject with a therapeutically effective amount of an FTI orselecting a cancer patient for an FTI treatment, wherein the 2DS5/2DL5ratio in a sample from the subject or cancer patient is higher than areference 2DS5/2DL5 ratio, and the subject or cancer patient meets atleast one of the five above described conditions regarding individualexpression level of the biomarkers. In some embodiments, the methodincludes treating a subject with a therapeutically effective amount ofan FTI or selecting a cancer patient for an FTI treatment, wherein the2DS2/2DL2 ratio in a sample from the subject or cancer patient is higherthan a reference 2DS2/2DL2 ratio, and 2DS5/2DL5 ratio in a sample fromthe subject or cancer patient is higher than a reference 2DS5/2DL5ratio, and the subject or cancer patient meets at least one of the fiveabove described conditions regarding individual expression level of thebiomarkers.

In some embodiments, the expression level of KIR2DL5 is the totalexpression levels of KIR2DL5A and KIR2DL5B. In some embodiments, theexpression level of KIR2DL5 is the expression level of KIR2DL5A. In someembodiments, the expression level of KIR2DL5 is the expression level ofKIR2DL5B. Thus, in some embodiments, the 2DS5/2DL5 ratio is the ratio ofexpression level KIR2DS5 to the total expression levels of KIR2DL5A andKIR2DL5B. In some embodiments, the 2DS5/2DL5 ratio is the ratio ofexpression level KIR2DS5 to the expression level of KIR2DL5A. In someembodiments, the 2DS5/2DL5 ratio is the ratio of expression levelKIR2DS5 to the expression level of KIR2DL5B.

To reiterate, the five conditions for selecting a subject or a cancerpatient for an FTI treatment based on expression level of singlebiomarker includes: condition 1: the expression level of KIR2DS2 in asample of the subject or cancer patient is higher than a referenceexpression level of KIR2DS2. condition 2: the expression level ofKIR2DS5 in a sample of the subject or cancer patient is higher than areference expression level of KIR2DS5; condition 3: the expression levelof KIR2DL2 in a sample of the subject or cancer patient is lower than areference expression level of KIR2DL2; condition 4: the expression levelof KIR2DL5 in a sample of the subject or cancer patient is lower than areference expression level of KIR2DL5; condition 5: the expression levelof GZMM in a sample of the subject or cancer patient is higher than areference expression level of GZMM. Any combination or permutation ofthese conditions can be used in further combination with either one orboth of the criteria based on expression ratio (the 2DS2/2DL2 ratio and2DS2/2DL2 ratio) as criteria for patient selection for an FTI treatment.

For example, in some embodiments, the method provided herein includestreating a subject with a therapeutically effective amount of an FTI orselecting a cancer patient for an FTI treatment, wherein the 2DS2/2DL2ratio in a sample from the subject or cancer patient is higher than areference 2DS2/2DL2 ratio, and the expression level of GZMM in a sampleof the subject is higher than a reference expression level of GZMM. Insome embodiments, the method includes treating a subject with atherapeutically effective amount of an FTI or selecting a cancer patientfor an FTI treatment, wherein the 2DS5/2DL5 ratio in a sample from thesubject or cancer patient is higher than a reference 2DS5/2DL5 ratio,and the expression level of KIR2DS2 in a sample of the subject is higherthan a reference expression level of KIR2DS2. A person of ordinary skillin the art would understand that the scope of the present invention isnot limited to these exemplary combinations and includes anycombinations or permutations of the individual criterion of patientselection for an FTI treatment as described herein.

As disclosed herein, the KIR genotype, HLA genotype, and the expressionprofile of some NK cell related genes can be used to predict theresponsiveness of a cancer patient to an FTI treatment. Accordingly,provided herein are methods for selecting cancer patients for an FTItreatment, or methods to treat a patient with FTI including first KIRtyping the patient or determining the expression profile of thebiomarkers identified herein to assess whether the patient is likely torespond to the treatment. The methods can further include HLA typing thepatient. A person of ordinary skill in the art would understand thatthese methods can be used independently as patient selection criteriafor an FTI treatment. Additionally, the methods can also be used inconnection with other patient stratification approaches to furtherincrease the response rate of a patient population to an FTI treatment.For example, in some embodiments, the methods provided herein furtherinclude determining the mutation status of the ras gene and selecting apatient for an FTI treatment when the patient has particular rasmutation, such as K-ras mutation, N-ras mutation, or H-ras mutation, asdescribed in greater detail below. In some embodiments, the methodsprovided herein further include determining the mutation status of theras gene and selecting a patient for an FTI treatment when the patienthas wild type K-ras and wild type N-ras. In some embodiments, themethods provided herein further include determining the mutation statusof the ras gene and selecting a patient for an FTI treatment when thepatient has a H-ras mutation. In other embodiments, the methods providedherein can further include using the 2 gene ratio between RASGRP1 andAPTX as additional patient selection criterion for an FTI treatment(U.S. Pat. No. 7,932,036, which is hereby incorporated by reference inits entirety). Methods described herein or otherwise known in the artcan be used to determine the mutation status of the ras gene orexpression of other biomarkers such as RASGRP1 or APTX. In someembodiments, the mutation status of a ras gene, such as H-ras, can bedetermined by NGS.

In some embodiments, the methods provided herein include determining theexpression level of a biomarker. In some embodiments, the expressionlevel of a biomarker can be the protein level of the biomarker. In someembodiments, the expression level of a biomarker can be the RNA level ofthe biomarker. Any method as described herein or otherwise known in theart to determine the protein level or RNA level of a gene can be usedfor determining the expression level of a biomarker in presentinvention.

Exemplary methods of detecting or quantitating mRNA levels include butare not limited to PCR-based methods, northern blots, ribonucleaseprotection assays, and the like. The mRNA sequence (e.g., the mRNA of abiomarker, such as CRBN or a CAP, or a fragment thereof) can be used toprepare a probe that is at least partially complementary. The probe canthen be used to detect the mRNA sequence in a sample, using any suitableassay, such as PCR-based methods, Northern blotting, a dipstick assay,and the like.

The commonly used methods known in the art for the quantification ofmRNA expression in a sample include northern blotting and in situhybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283(1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992));and polymerase chain reaction (PCR) (Weis et ah, Trends in Genetics8:263-264 (1992)). Alternatively, antibodies may be employed that canrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methodsfor sequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), and gene expression analysis by massivelyparallel signature sequencing (MPSS).

A sensitive and flexible quantitative method is PCR. Examples of PCRmethods can be found in the literature. Examples of PCR assays can befound in U.S. Pat. No. 6,927,024, which is incorporated by referenceherein in its entirety. Examples of RT-PCR methods can be found in U.S.Pat. No. 7,122,799, which is incorporated by reference herein in itsentirety. A method of fluorescent in situ PCR is described in U.S. Pat.No. 7,186,507, which is incorporated by reference herein in itsentirety.

It is noted, however, that other nucleic acid amplification protocols(i.e., other than PCR) may also be used in the nucleic acid analyticalmethods described herein. For example, suitable amplification methodsinclude ligase chain reaction (see, e.g., Wu & Wallace, Genomics4:560-569, 1988); strand displacement assay (see, e.g., Walker et al.,Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Pat. No. 5,455,166);and several transcription-based amplification systems, including themethods described in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491;the transcription amplification system (TAS) (Kwoh et al., Proc. Natl.Acad. Sci. USA 86: 1173-1177, 1989); and self-sustained sequencereplication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; WO 92/08800). Alternatively, methods that amplify theprobe to detectable levels can be used, such as Q-replicaseamplification (Kramer & Lizardi, Nature 339:401-402, 1989; Lomeli etal., Clin. Chem. 35: 1826-1831, 1989). A review of known amplificationmethods is provided, for example, by Abramson and Myers in CurrentOpinion in Biotechnology 4:41-47 (1993).

mRNA may be isolated from the starting tissue sample. General methodsfor mRNA extraction are well known in the art and are disclosed instandard textbooks of molecular biology, including Ausubel et al.,Current Protocols of Molecular Biology, John Wiley and Sons (1997). Inparticular, RNA isolation can be performed using purification kit,buffer set and protease from commercial manufacturers, such as Qiagen,according to the manufacturer's instructions. For example, total RNAfrom cells in culture can be isolated using Qiagen RNeasy mini-columns.Other commercially available RNA isolation kits include MASTERPURE®Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), andParaffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissuesamples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared fromtumor can be isolated, for example, by cesium chloride density gradientcentrifugation.

In some embodiments, the first step in gene expression profiling by PCRis the reverse transcription of the RNA template into cDNA, followed byits exponential amplification in a PCR reaction. In other embodiments, acombined reverse-transcription-polymerase chain reaction (RT-PCR)reaction may be used, e.g., as described in U.S. Pat. Nos. 5,310,652;5,322,770; 5,561,058; 5,641,864; and 5,693,517. The two commonly usedreverse transcriptases are avilo myeloblastosis virus reversetranscriptase (AMV-RT) and Moloney murine leukemia virus reversetranscriptase (MMLV-RT). The reverse transcription step is typicallyprimed using specific primers, random hexamers, or oligo-dT primers,depending on the circumstances and the goal of expression profiling. Forexample, extracted RNA can be reverse-transcribed using a GENEAMP™ RNAPCR kit (Perkin Elmer, Calif, USA), following the manufacturer'sinstructions. The derived cDNA can then be used as a template in thesubsequent PCR reaction.

In some embodiments, Real-Time Reverse Transcription-PCR (qRT-PCR) canbe used for both the detection and quantification of RNA targets(Bustin, et al., 2005, Clin. Sci., 109:365-379). Examples ofqRT-PCR-based methods can be found, for example, in U.S. Pat. No.7,101,663, which is incorporated by reference herein in its entirety.Instruments for real-time PCR, such as the Applied Biosystems 7500, areavailable commercially, as are the reagents, such as TaqMan SequenceDetection chemistry.

For example, TaqMan® Gene Expression Assays can be used, following themanufacturer's instructions. These kits are pre-formulated geneexpression assays for rapid, reliable detection and quantification ofhuman, mouse and rat mRNA transcripts. TaqMan® or 5′-nuclease assay, asdescribed in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; andHolland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280, can beused. TAQMAN® PCR typically utilizes the 5′-nuclease activity of Taq orTth polymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

Any method suitable for detecting degradation product can be used in a5′ nuclease assay. Often, the detection probe is labeled with twofluorescent dyes, one of which is capable of quenching the fluorescenceof the other dye. The dyes are attached to the probe, preferably oneattached to the 5′ terminus and the other is attached to an internalsite, such that quenching occurs when the probe is in an unhybridizedstate and such that cleavage of the probe by the 5′ to 3′ exonucleaseactivity of the DNA polymerase occurs in between the two dyes.

Amplification results in cleavage of the probe between the dyes with aconcomitant elimination of quenching and an increase in the fluorescenceobservable from the initially quenched dye. The accumulation ofdegradation product is monitored by measuring the increase in reactionfluorescence. U.S. Pat. Nos. 5,491,063 and 5,571,673, both incorporatedherein by reference, describe alternative methods for detecting thedegradation of probe which occurs concomitant with amplification.5′-Nuclease assay data may be initially expressed as Ct, or thethreshold cycle. As discussed above, fluorescence values are recordedduring every cycle and represent the amount of product amplified to thatpoint in the amplification reaction. The point when the fluorescentsignal is first recorded as statistically significant is the thresholdcycle (Ct).

To minimize errors and the effect of sample-to-sample variation, PCR isusually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and P-actin.

PCR primers and probes are designed based upon intron sequences presentin the gene to be amplified. In this embodiment, the first step in theprimer/probe design is the delineation of intron sequences within thegenes. This can be done by publicly available software, such as the DNABLAT software developed by Kent, W., Genome Res. 12(4):656-64 (2002), orby the BLAST software including its variations. Subsequent steps followwell established methods of PCR primer and probe design.

In order to avoid non-specific signals, it can be important to maskrepetitive sequences within the introns when designing the primers andprobes. This can be easily accomplished by using the Repeat Maskerprogram available on-line through the Baylor College of Medicine, whichscreens DNA sequences against a library of repetitive elements andreturns a query sequence in which the repetitive elements are masked.The masked intron sequences can then be used to design primer and probesequences using any commercially or otherwise publicly availableprimer/probe design packages, such as Primer Express (AppliedBiosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Rozenand Skaletsky (2000) Primer3 on the WWW for general users and forbiologist programmers. In: Krawetz S, Misener S (eds) BioinformaticsMethods and Protocols: Methods in Molecular Biology. Humana Press,Totowa, N.J., pp 365-386).

Factors considered in PCR primer design include primer length, meltingtemperature (Tm), and G/C content, specificity, complementary primersequences, and 3′-end sequence. In general, optimal PCR primers aregenerally 17-30 bases in length, and contain about 20-80%, such as, forexample, about 50-60% G+C bases. Tm's between 50 and 80° C., e.g. about50 to 70° C. are typically preferred. For further guidelines for PCRprimer and probe design see, e.g. Dieffenbach et ah, “General Conceptsfor PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand,“Optimization of PCRs” in: PCR Protocols, A Guide to Methods andApplications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N.Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527(1997), the entire disclosures of which are hereby expresslyincorporated by reference.

An exemplary PCR program, for example, is 50° C. for 2 minutes, 95° C.for 10 minutes, 40 cycles of 95° C. for 15 seconds, then 60° C. for 1minute. To determine the cycle number at which the fluorescence signalassociated with a particular amplicon accumulation crosses the threshold(referred to as the CT), the data can be analyzed, for example, using a7500 Real-Time PCR System Sequence Detection software v1.3 using thecomparative CT relative quantification calculation method. Using thismethod, the output is expressed as a fold-change of expression levels.In some embodiments, the threshold level can be selected to beautomatically determined by the software. In some embodiments, thethreshold level is set to be above the baseline but sufficiently low tobe within the exponential growth region of an amplification curve.

RNA-Seq, also called Whole Transcriptome Shotgun Sequencing (WTSS)refers to the use of high-throughput sequencing technologies to sequencecDNA in order to get information about a sample's RNA content.Publications describing RNA-Seq include: Wang et al., Nature ReviewsGenetics 10 (1): 57-63 (January 2009); Ryan et al. BioTechniques 45 (1):81-94 (2008); and Maher et al., Nature 458 (7234): 97-101 (January2009); which are hereby incorporated in their entirety.

Differential gene expression can also be identified, or confirmed usingthe microarray technique. In this method, polynucleotide sequences ofinterest (including cDNAs and oligonucleotides) are plated, or arrayed,on a microchip substrate. The arrayed sequences are then hybridized withspecific DNA probes from cells or tissues of interest.

In an embodiment of the microarray technique, PCR amplified inserts ofcDNA clones are applied to a substrate in a dense array. Preferably atleast 10,000 nucleotide sequences are applied to the substrate. Themicroarrayed genes, immobilized on the microchip at 10,000 elementseach, are suitable for hybridization under stringent conditions.Fluorescently labeled cDNA probes may be generated through incorporationof fluorescent nucleotides by reverse transcription of RNA extractedfrom tissues of interest. Labeled cDNA probes applied to the chiphybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement allows for assessment of corresponding mRNA abundance. With dualcolor fluorescence, separately labeled cDNA probes generated from twosources of RNA are hybridized pairwise to the array. The relativeabundance of the transcripts from the two sources corresponding to eachspecified gene is thus determined simultaneously. The miniaturized scaleof the hybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al., Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)).Microarray analysis can be performed by commercially availableequipment, following manufacturer's protocols, such as by using theAffymetrix GENCHIP™ technology, or Incyte's microarray technology.

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. First, a short sequence tag (about 10-14 bp)is generated that contains sufficient information to uniquely identify atranscript, provided that the tag is obtained from a unique positionwithin each transcript. Then, many transcripts are linked together toform long serial molecules, that can be sequenced, revealing theidentity of the multiple tags simultaneously. The expression pattern ofany population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. For more details see, e.g. Velculescu et ah,Science 270:484-487 (1995); and Velculescu et al, Cell 88:243-51 (1997).

The MassARRAY (Sequenom, San Diego, Calif.) technology is an automated,high-throughput method of gene expression analysis using massspectrometry (MS) for detection. According to this method, following theisolation of RNA, reverse transcription and PCR amplification, the cDNAsare subjected to primer extension. The cDNA-derived primer extensionproducts are purified, and dispensed on a chip array that is pre-loadedwith the components needed for MALTI-TOF MS sample preparation. Thevarious cDNAs present in the reaction are quantitated by analyzing thepeak areas in the mass spectrum obtained.

mRNA level can also be measured by an assay based on hybridization. Atypical mRNA assay method can contain the steps of 1) obtainingsurface-bound subject probes; 2) hybridization of a population of mRNAsto the surface-bound probes under conditions sufficient to provide forspecific binding (3) post-hybridization washes to remove nucleic acidsnot bound in the hybridization; and (4) detection of the hybridizedmRNAs. The reagents used in each of these steps and their conditions foruse may vary depending on the particular application.

Any suitable assay platform can be used to determine the mRNA level in asample. For example, an assay can be in the form of a dipstick, amembrane, a chip, a disk, a test strip, a filter, a microsphere, aslide, a multiwell plate, or an optical fiber. An assay system can havea solid support on which a nucleic acid corresponding to the mRNA isattached. The solid support can have, for example, a plastic, silicon, ametal, a resin, glass, a membrane, a particle, a precipitate, a gel, apolymer, a sheet, a sphere, a polysaccharide, a capillary, a film aplate, or a slide. The assay components can be prepared and packagedtogether as a kit for detecting an mRNA.

The nucleic acid can be labeled, if desired, to make a population oflabeled mRNAs. In general, a sample can be labeled using methods thatare well known in the art (e.g., using DNA ligase, terminal transferase,or by labeling the RNA backbone, etc.; see, e.g., Ausubel, et al., ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 ColdSpring Harbor, N.Y.). In some embodiments, the sample is labeled withfluorescent label. Exemplary fluorescent dyes include but are notlimited to xanthene dyes, fluorescein dyes, rhodamine dyes, fluoresceinisothiocyanate (FITC), 6 carboxyfluorescein (FAM), 6carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 6 carboxy 4′, 5′dichloro 2′, 7′ dimethoxyfluorescein (JOE or J), N,N,N′,N′ tetramethyl 6carboxyrhodamine (TAMRA or T), 6 carboxy X rhodamine (ROX or R), 5carboxyrhodamine 6G (R6G5 or G5), 6 carboxyrhodamine 6G (R6G6 or G6),and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; Alexa dyes,e.g. Alexa-fluor-555; coumarin, Diethylaminocoumarin, umbelliferone;benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red;ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes;porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, Pyrene,Fluorescein Chlorotriazinyl, R110, Eosin, JOE, R6G,Tetramethylrhodamine, Lissamine, ROX, Napthofluorescein, and the like.

Hybridization can be carried out under suitable hybridizationconditions, which may vary in stringency as desired. Typical conditionsare sufficient to produce probe/target complexes on a solid surfacebetween complementary binding members, i.e., between surface-boundsubject probes and complementary mRNAs in a sample. In certainembodiments, stringent hybridization conditions can be employed.

Hybridization is typically performed under stringent hybridizationconditions. Standard hybridization techniques (e.g. under conditionssufficient to provide for specific binding of target mRNAs in the sampleto the probes) are described in Kallioniemi et al., Science 258:818-821(1992) and WO 93/18186. Several guides to general techniques areavailable, e.g., Tijssen, Hybridization with Nucleic Acid Probes, PartsI and II (Elsevier, Amsterdam 1993). For descriptions of techniquessuitable for in situ hybridizations, see Gall et al. Meth. Enzymol.,21:470-480 (1981); and Angerer et al. in Genetic Engineering: Principlesand Methods (Setlow and Hollaender, Eds.) Vol 7, pgs 43-65 (PlenumPress, New York 1985). Selection of appropriate conditions, includingtemperature, salt concentration, polynucleotide concentration,hybridization time, stringency of washing conditions, and the like willdepend on experimental design, including source of sample, identity ofcapture agents, degree of complementarity expected, etc., and may bedetermined as a matter of routine experimentation for those of ordinaryskill in the art. Those of ordinary skill will readily recognize thatalternative but comparable hybridization and wash conditions can beutilized to provide conditions of similar stringency.

After the mRNA hybridization procedure, the surface boundpolynucleotides are typically washed to remove unbound nucleic acids.Washing may be performed using any convenient washing protocol, wherethe washing conditions are typically stringent, as described above. Thehybridization of the target mRNAs to the probes is then detected usingstandard techniques.

In some embodiments, the methods provided herein include determining themRNA level of one or more biomarkers selected from the group consistingof KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM using one of theapproaches described herein or otherwise known in the art. In oneembodiment, the methods include determining the KIR2DS2 mRNA level froma sample of a subject. In one embodiment, the methods includedetermining the KIR2DL2 mRNA level from a sample of a subject. In oneembodiment, the methods include determining the KIR2DS5 mRNA level froma sample of a subject. In one embodiment, the methods includedetermining the KIR2DL5 mRNA level from a sample of a subject. In oneembodiment, the methods include determining the GZMM mRNA level from asample of a subject.

In some embodiments, the mRNA level of KIR2DL5 is the total mRNA levelsof KIR2DL5A and KIR2DL5B. In some embodiments, the mRNA level of KIR2DL5is the mRNA level of KIR2DL5A. In some embodiments, the mRNA level ofKIR2DL5 is the mRNA level of KIR2DL5B.

In some embodiments, the methods provided herein include determining themRNA levels of two or more of biomarkers selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM. In someembodiments, the methods include determining the mRNA levels of three ormore of these biomarkers. In some embodiments, the methods includedetermining the mRNA levels of four or more of these biomarkers. In someembodiments, the methods include determining the mRNA levels of all fiveof these biomarkers.

In some embodiments, the methods provided herein include determiningmRNA levels of KIR2DS2 and KIR2DL2 in a sample from a subject or acancer patient, and calculating the ratio of mRNA level of KIR2DS2 tomRNA level KIR2DL2 (the 2DS2/2DL2 mRNA ratio). In some embodiments, themethods further include determining mRNA levels of KIR2DS5 and KIR2DL5 asample from a subject or a cancer patient, and calculating the ratio ofmRNA level of KIR2DS5 to mRNA level KIR2DL5 (the 2DS5/2DL5 mRNA ratio).In some embodiments, the methods further include determining mRNA levelsof KIR2DS2 and KIR2DL2 in a sample from a subject or a cancer patient,and mRNA levels of KIR2DS5 and KIR2DL5 a sample from a subject or acancer patient, and calculating both the 2DS2/2DL2 mRNA ratio and the2DS5/2DL5 mRNA ratio.

In some embodiments, the mRNA level of one or more biomarkers selectedfrom the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, andGZMM is determined by qPCR, RT-PCR, RNA-seq, microarray analysis, SAGE,MassARRAY technique, or FISH. In some embodiments, the mRNA level of oneor more of the biomarkers selected from the group consisting of KIR2DS2,KIR2DL2, KIR2DS5, KIR2DL5, and GZMM is determined by qPCR or RT-PCR.

In some embodiments, the methods provided herein include determining theprotein level of one or more biomarkers selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM. The proteinlevel of the biomarker can be detected by a variety ofimmunohistochemistry (IHC) approaches or other immunoassay methods.

IHC staining of tissue sections has been shown to be a reliable methodof assessing or detecting presence of proteins in a sample.Immunohistochemistry techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. Thus, antibodies or antisera, preferably polyclonalantisera, and most preferably monoclonal antibodies specific for eachmarker are used to detect expression. As discussed in greater detailbelow, the antibodies can be detected by direct labeling of theantibodies themselves, for example, with radioactive labels, fluorescentlabels, hapten labels such as, biotin, or an enzyme such as horse radishperoxidase or alkaline phosphatase. Alternatively, unlabeled primaryantibody is used in conjunction with a labeled secondary antibody,comprising antisera, polyclonal antisera or a monoclonal antibodyspecific for the primary antibody. Immunohistochemistry protocols andkits are well known in the art and are commercially available. Automatedsystems for slide preparation and IHC processing are availablecommercially. The Ventana® BenchMark XT system is an example of such anautomated system.

Standard immunological and immunoassay procedures can be found in Basicand Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover,the immunoassays can be performed in any of several configurations,which are reviewed extensively in Enzyme Immunoassay (Maggio, ed.,1980); and Harlow & Lane, supra. For a review of the generalimmunoassays, see also Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Ten, eds., 7th ed. 1991).

Commonly used assays to detect protein level of a biomarker includenoncompetitive assays, e.g., sandwich assays, and competitive assays.Typically, an assay such as an ELISA assay can be used. ELISA assays areknown in the art, e.g., for assaying a wide variety of tissues andsamples, including blood, plasma, serum or bone marrow.

A wide range of immunoassay techniques using such an assay format areavailable, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and4,018,653, which are hereby incorporated by reference in theirentireties. These include both single-site and two-site or “sandwich”assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. These assays also include direct binding ofa labeled antibody to a target biomarker. Sandwich assays are commonlyused assays. A number of variations of the sandwich assay techniqueexist. For example, in a typical forward assay, an unlabelled antibodyis immobilized on a solid substrate, and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen complex, a second antibody specific to the antigen,labeled with a reporter molecule capable of producing a detectablesignal is then added and incubated, allowing time sufficient for theformation of another complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal produced by the reporter molecule.The results may either be qualitative, by simple observation of thevisible signal, or may be quantitated by comparing with a control samplecontaining known amounts of biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labeled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface may be glass or a polymer, the most commonly usedpolymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride, or polypropylene. The solid supports may be in the form oftubes, beads, discs of microplates, or any other surface suitable forconducting an immunoassay. The binding processes are well-known in theart and generally consist of cross-linking covalently binding orphysically adsorbing, the polymer-antibody complex is washed inpreparation for the test sample. An aliquot of the sample to be testedis then added to the solid phase complex and incubated for a period oftime sufficient (e.g. 2-40 minutes or overnight if more convenient) andunder suitable conditions (e.g., from room temperature to 40° C. such asbetween 25° C. and 32° C. inclusive) to allow binding of any subunitpresent in the antibody. Following the incubation period, the antibodysubunit solid phase is washed and dried and incubated with a secondantibody specific for a portion of the biomarker. The second antibody islinked to a reporter molecule which is used to indicate the binding ofthe second antibody to the molecular marker.

In some embodiments, flow cytometry (FACS) can be used to detect theprotein level of a biomarker. Surface proteins (such as KIRs) can bedetected using antibodies against specific biomarkers. The flowcytometer detects and reports the intensity of the fluorichrome-taggedantibody, which indicates the expression level of the biomarker.Non-fluorescent cytoplasmic proteins can also be observed by stainingpermeablized cells. The stain can either be a fluorescence compound ableto bind to certain molecules, or a fluorichrome-tagged antibody to bindthe molecule of choice.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibody,which may or may not be labeled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labeling with the antibody.Alternatively, a second labeled antibody, specific to the first antibodyis exposed to the target-first antibody complex to form a target-firstantibody-second antibody tertiary complex. The complex is detected bythe signal emitted by a labeled reporter molecule.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase, and alkaline phosphatase, and other arediscussed herein. The substrates to be used with the specific enzymesare generally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. Examples of suitableenzymes include alkaline phosphatase and peroxidase. It is also possibleto employ fluorogenic substrates, which yield a fluorescent productrather than the chromogenic substrates noted above. In all cases, theenzyme-labeled antibody is added to the first antibody-molecular markercomplex, allowed to bind, and then the excess reagent is washed away. Asolution containing the appropriate substrate is then added to thecomplex of antibody-antigen-antibody. The substrate will react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of biomarkerwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein and rhodamine, can be chemically coupled toantibodies without altering their binding capacity. When activated byillumination with light of a particular wavelength, thefluorochrome-labeled antibody adsorbs the light energy, inducing a stateto excitability in the molecule, followed by emission of the light at acharacteristic color visually detectable with a light microscope. As inthe EIA, the fluorescent labeled antibody is allowed to bind to thefirst antibody-molecular marker complex. After washing off the unboundreagent, the remaining tertiary complex is then exposed to the light ofthe appropriate wavelength, the fluorescence observed indicates thepresence of the molecular marker of interest. Immunofluorescence and EIAtechniques are both very well established in the art and are discussedherein.

In some embodiments, the methods provided herein include determining theprotein level of one or more biomarkers selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a samplefrom a subject or a cancer patient using one of the approaches describedherein or otherwise known in the art. In one embodiment, the methodincludes determining the KIR2DS2 protein level from a sample of asubject or a cancer patient. In one embodiment, the methods of presentinvention includes determining the KIR2DL2 protein level from a sampleof a subject or a cancer patient. In one embodiment, the method includesdetermining the KIR2DS5 protein level from a sample of a subject or acancer patient. In one embodiment, the method includes determining theKIR2DL5 protein level from a sample of a subject or a cancer patient. Inone embodiment, the method includes determining the GZMM protein levelfrom a sample of a subject or a cancer patient.

In some embodiments, the protein level of KIR2DL5 is the total proteinlevels of KIR2DL5A and KIR2DL5B. In some embodiments, the protein levelof KIR2DL5 is the protein level of KIR2DL5A. In some embodiments, theprotein level of KIR2DL5 is the protein level of KIR2DL5B.

In some embodiments, the methods provided herein include determining theprotein levels of two or more of these biomarkers. In some embodiments,the methods include determining the protein levels of three or more ofthese biomarkers. In some embodiments, the methods include determiningthe protein levels of four or more of these biomarkers. In someembodiments, the methods include determining the protein levels of fiveof these biomarkers.

In some embodiments, the methods provided herein further includedetermining protein levels of KIR2DS2 and KIR2DL2 in a sample from asubject or a cancer patient, and calculating the ratio of protein levelof KIR2DS2 to protein level KIR2DL2 (the 2DS2/2DL2 protein ratio). Insome embodiments, the methods further include determining protein levelsof KIR2DS5 and KIR2DL5 a sample from a subject or a cancer patient, andcalculating the ratio of protein level of KIR2DS5 to protein levelKIR2DL5 (the 2DS5/2DL5 protein ratio). In some embodiments, the methodsfurther include determining protein levels of KIR2DS2 and KIR2DL2 in asample from a subject or a cancer patient, and protein levels of KIR2DS5and KIR2DL5 a sample from a subject or a cancer patient, and calculatingboth the 2DS2/2DL2 protein ratio and the 2DS5/2DL5 protein ratio.

In some embodiments, the protein level of one or more biomarkersselected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5,KIR2DL5, and GZMM is determined by immunoblotting (Western blot), ELISA,immunohistochemistry, flow cytometry, cytometric bead array or massspectroscopy. In some embodiments, the protein level of one or morebiomarkers selected from the group consisting of KIR2DS2, KIR2DL2,KIR2DS5, KIR2DL5, and GZMM is determined by ELISA.

3.3. SAMPLES

In some embodiments, the methods of KIR typing, HLA typing or themethods of determining either the mRNA level or the protein level of oneor more biomarkers further include obtaining a sample from a subject.The subject can be a mammal, for example, a human. The subject can bemale or female, and can be an adult, child or infant. The subject can bea patient. The patient can be a cancer patient. Samples can be analyzedat a time during an active phase of a cancer (e.g., lymphoma, MDS, orleukemia), or when the cancer is inactive. In certain embodiments, morethan one sample from a subject can be obtained.

In some embodiments, the methods of KIR typing, HLA typing or themethods of determining either the mRNA level or the protein level of oneor more biomarkers is performed as a companion diagnostic to the FTItreatment. The companion diagnostic can be performed at the clinic sitewhere the subject is treated. The companion diagnostic can also beperformed at a site separate from the clinic site where the subject istreated.

In certain embodiments, the sample used in the methods provided hereinincludes body fluids from a subject. Non-limiting examples of bodyfluids include blood (e.g., peripheral whole blood, peripheral blood),blood plasma, bone marrow, amniotic fluid, aqueous humor, bile, lymph,menses, serum, urine, cerebrospinal fluid surrounding the brain and thespinal cord, synovial fluid surrounding bone joints.

In one embodiment, the sample is a bone marrow sample. Procedures toobtain a bone marrow sample are well known in the art, including but notlimited to bone marrow biopsy and bone marrow aspiration. Bone marrowhas a fluid portion and a more solid portion. In bone marrow biopsy, asample of the solid portion is taken. In bone marrow aspiration, asample of the fluid portion is taken. Bone marrow biopsy and bone marrowaspiration can be done at the same time and referred to as a bone marrowexam.

In some embodiments, the sample is a blood sample. The blood sample canbe obtained using conventional techniques as described in, e.g. Innis etal, editors, PCR Protocols (Academic Press, 1990). White blood cells canbe separated from blood samples using convention techniques orcommercially available kits, e.g. RosetteSep kit (Stein CellTechnologies, Vancouver, Canada). Sub-populations of white blood cells,e.g. mononuclear cells, NK cells, B cells, T cells, monocytes,granulocytes or lymphocytes, can be further isolated using conventionaltechniques, e.g. magnetically activated cell sorting (MACS) (MiltenyiBiotec, Auburn, Calif.) or fluorescently activated cell sorting (FACS)(Becton Dickinson, San Jose, Calif.).

In one embodiment, the blood sample is from about 0.1 mL to about 10.0mL, from about 0.2 mL to about 7 mL, from about 0.3 mL to about 5 mL,from about 0.4 mL to about 3.5 mL, or from about 0.5 mL to about 3 mL.In another embodiment, the blood sample is about 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0,8.0, 9.0 or 10.0 mL.

In some embodiments, the sample used in the present methods includes abiopsy (e.g., a tumor biopsy). The biopsy can be from any organ ortissue, for example, skin, liver, lung, heart, colon, kidney, bonemarrow, teeth, lymph node, hair, spleen, brain, breast, or other organs.Any biopsy technique known by those skilled in the art can be used forisolating a sample from a subject, for instance, open biopsy, closebiopsy, core biopsy, incisional biopsy, excisional biopsy, or fineneedle aspiration biopsy.

In one embodiment, the sample used in the methods provided herein isobtained from the subject prior to the subject receiving a treatment forthe disease or disorder. In another embodiment, the sample is obtainedfrom the subject during the subject receiving a treatment for thedisease or disorder. In another embodiment, the sample is obtained fromthe subject after the subject receiving a treatment for the disease ordisorder. In various embodiments, the treatment includes administeringan FTI to the subject.

In certain embodiments, the sample used in the methods provided hereinincludes a plurality of cells. Such cells can include any type of cells,e.g., stem cells, blood cells (e.g., PBMCs), lymphocytes, NK cells, Bcells, T cells, monocytes, granulocytes, immune cells, or tumor orcancer cells. In certain embodiments, the sample used in the methodsprovided herein includes a plurality of enriched NK cells from a bloodsample or a bone marrow sample of a subject, or a cancer patient.Specific cell populations can be obtained using a combination ofcommercially available antibodies (e.g., Quest Diagnostic (San JuanCapistrano, Calif.); Dako (Denmark)).

In certain embodiments, the sample used in the methods provided hereinis from a diseased tissue, e.g., from an individual having cancer (e.g.,lymphoma, MDS, or leukemia). In certain embodiments. In someembodiments, the cells can be obtained from the tumor or cancer cells ora tumor tissue, such as a tumor biopsy or a tumor explants. In certainembodiments, the number of cells used in the methods provided herein canrange from a single cell to about 10⁹ cells. In some embodiments, thenumber of cells used in the methods provided herein is about 1×10⁴,5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, or 5×10⁸.

The number and type of cells collected from a subject can be monitored,for example, by measuring changes in morphology and cell surface markersusing standard cell detection techniques such as flow cytometry, cellsorting, immunocytochemistry (e.g., staining with tissue specific orcell-marker specific antibodies) fluorescence activated cell sorting(FACS), magnetic activated cell sorting (MACS), by examination of themorphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. Fluorescence activated cell sorting (FACS) is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In certain embodiments, subsets of cells are used in the methodsprovided herein. Methods to sort and isolate specific populations ofcells are well-known in the art and can be based on cell size,morphology, or intracellular or extracellular markers. Such methodsinclude, but are not limited to, flow cytometry, flow sorting, FACS,bead based separation such as magnetic cell sorting, size-basedseparation (e.g., a sieve, an array of obstacles, or a filter), sortingin a microfluidics device, antibody-based separation, sedimentation,affinity adsorption, affinity extraction, density gradientcentrifugation, laser capture microdissection, etc. In some embodiments,the sample include enriched NK cells sorted by one or more methodsdescribed herein, or otherwise known in the art. In one embodiments, theenriched NK cells are further expanded in vitro before being subjectedto KIR typing, HLA typing or analysis of expression level of one or morebiomarkers selected from the group consisting of KIR2DS2, KIR2DL2,KIR2DS5, KIR2DL5 and GZMM.

The sample can be a whole blood sample, a bone marrow sample, apartially purified blood sample, PBMCs, or tissue biopsy. In someembodiments, the sample is a bone marrow sample from a cancer patient.In some embodiments, the sample is PBMCs from a cancer patient. In someembodiments, the sample is enriched NK cells from bone marrow, wholeblood, or partially purified blood. In some embodiments, the NK cellsare further expanded in vitro. Methods of obtaining a sample from asubject and methods to prepare the sample for determining either themRNA level or the protein level of one or more biomarkers are well knownin the art.

3.4. REFERENCE LEVELS AND REFERENCE RATIOS

Provided herein are methods of treating a subject with a therapeuticallyeffective amount of an FTI or selecting a cancer patient for an FTItreatment, based on KIR typing, expression level (either mRNA level orprotein level) of one or more of individual biomarkers selected from thegroup consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, oreither or both of the ratio of expression levels between biomarkers (the2DS2/2DL2 ratio; the 2DS5/2DL5 ratio). In some embodiments, a cancerpatient is selected for FTI treatment if the cancer patient is a carrierof KIR2DS2 or KIR2DS5, or both. In some embodiments, a cancer patient isselected for FTI treatment if

(i) the expression level of KIR2DS2 in a sample from the subject ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the subject islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the subject ishigher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the subject islower than a reference expression level of KIR2DL5;

(v) the expression level of GZMM in a sample from the subject is higherthan a reference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in a sample from the subject is higher than a referenceratio of an expression level of KIR2DS2 to an expression level ofKIR2DL2; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in a sample from the subject is higher than a referenceratio of an expression level of KIR2DS5 to an expression level ofKIR2DL5; or any combination of (i)-(vii).

The methods provided herein can further include determining a referenceexpression level of each individual biomarker selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, or thereference ratio between expression level of biomarkers, including thereference 2DS2/2DL2 ratio and the reference 2DS5/2DL5 ratio. In someembodiments, the reference expression level of a biomarker is theexpression level of the biomarker in a sample from a healthy individual,or the average or median expression level of the biomarker in multiplesamples from one or multiple healthy individuals. In some embodiments,the reference expression level of a biomarker is the average expressionlevel of the biomarker in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50or more healthy individuals. In some embodiments, the referenceexpression level of a biomarker is the medium expression level of thebiomarker in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50 or morehealthy individuals.

In some embodiments, the reference expression level of KIR2DS2 is theexpression level of KIR2DS2 in a sample from a healthy individual, orthe average or median expression level of the KIR2DS2 in multiplesamples from one or multiple healthy individuals. In some embodiments,the reference expression level of KIR2DL2 is the expression level ofKIR2DL2 in a sample from a healthy individual, or the average or medianexpression level of the KIR2DL2 in multiple samples from one or multiplehealthy individuals. In some embodiments, the reference expression levelof KIR2DS5 is the expression level of KIR2DS5 in a sample from a healthyindividual, or the average or median expression level of the KIR2DS5 inmultiple samples from one or multiple healthy individuals. In someembodiments, the reference expression level of KIR2DL5 is the expressionlevel of KIR2DL5 in a sample from a healthy individual, or the averageor median expression level of the KIR2DL5 in multiple samples from oneor multiple healthy individuals. In some embodiments, the referenceexpression level of GZMM is the expression level of GZMM in a samplefrom a healthy individual, or the average or median expression level ofthe GZMM in multiple samples from one or multiple healthy individuals.

In some embodiments, methods provided herein further include determininga reference ratio between expression levels of two biomarkers, such asthe 2DS2/2DL2 reference expression ratio, or the 2DS5/2DL5 referenceexpression ratio. In some embodiments, the reference expression ratio ofbiomarkers is the expression ratio of the biomarkers in a sample from ahealthy individual, or the average or median expression ratio of thebiomarkers in multiple samples from one or multiple healthy individuals.In some embodiments, the reference expression ratio of two biomarkers isthe average expression ratio of the biomarkers in samples from 2, 3, 5,10, 15, 20, 30, 40, 50 or more healthy individuals. In some embodiments,the reference expression ratio of two biomarkers is the mediumexpression ratio of the biomarkers in samples from 2, 3, 5, 10, 15, 20,30, 40, 50 or more healthy individuals.

In some embodiments, the reference 2DS2/2DL2 ratio is the 2DS2/2DL2ratio in a sample from a healthy individual, or the average or median2DS2/2DL2 ratio in multiple samples from one or multiple healthyindividuals. In some embodiments, the reference 2DS5/2DL5 ratio is the2DS5/2DL5 in a sample from a healthy individual, or the average ormedian 2DS5/2DL5 in multiple samples from one or multiple healthyindividuals.

In some embodiments, the reference expression level of a biomarker orthe reference ratio between expression levels of two biomarkers can bedetermined based on statistical analysis of data from previous clinicaltrials, including outcome of a group of patients, namely, the patients'responsiveness to an FTI treatment, as well as the expression levels ofthe biomarker or ratio of expression levels between biomarkers of thegroup of patients. A number of statistical methods are well known in theart to determine the reference level (or referred to as the “cut-offvalue”) of one or more biomarkers when used to predict theresponsiveness of a patient to a particular treatment, or to stratifypatients for a particular treatment.

One method of the invention includes analyzing gene expression profilesfor biomarkers identified herein that distinguish responder fromnon-responder to determine the reference expression level for one ormore biomarkers. Comparisons between responders and non-responders canbe performed using the Mann-Whitney U-test, Chi-square test, or Fisher'sExact test. Analysis of descriptive statistics and comparisons can beperformed using SigmaStat Software (Systat Software, Inc., San Jose,Calif., USA).

In some embodiments, a classification and regression tree (CART)analysis can be adopted to determine the reference level. CART analysisis based on a binary recursive partitioning algorithm and allows for thediscovery of complex predictor variable interactions that may not beapparent with more traditional methods, such as multiple linearregression. Binary recursive partitioning refers to the analysis thatis: 1) binary, meaning there were two possible outcome variables, namely“responders”and “non-responders,” with the effect of splitting patientsinto 2 groups; 2) recursive, meaning the analysis can be performedmultiple times; and 3) partitioned, meaning the entire data set can besplit into sections. This analysis also has the ability to eliminatepredictor variables with poor performance. The classification tree canbe built using Salford Predictive Modeler v6.6 (Salford Systems, SanDiego, Calif., USA).

Articles of this invention are representations of the gene expressionprofiles useful for predicting the responsiveness of a cancer patient toan FTI treatment that are reduced to a medium that can be automaticallyread such as computer readable media (magnetic, optical, and the like).The articles can also include instructions for assessing the geneexpression profiles in such media. For example, the articles maycomprise a CD-ROM having computer instructions for comparing geneexpression profiles of biomarkers described above. The articles may alsohave gene expression profiles digitally recorded therein so that theymay be compared with gene expression data from patient samples.Alternatively, the profiles can be recorded in differentrepresentational format. Clustering algorithms such as thoseincorporated in “OMNIVIZ” and “TREE VIEW” computer programs mentionedabove can best assist in the visualization of such data.

Receiver Operator Characteristic (ROC) analysis can be utilized todetermine the reference expression level, or reference expression ratio,or test the overall predictive value of individual genes and/ormultigene classifiers. A review of the ROC analysis can be found inSoreide, J Clin Pathol 10.1136 (2008), which is hereby incorporated byreference in its entirety.

The reference level can be determined from the ROC curve of the trainingset to ensure both high sensitivity and high specificity. To determinehow many biomarkers are needed to be included in the predictor,leave-one-out cross validation (LOOCV) can be used. The response scoresfor the ‘left-out’ samples based on different numbers of genes arerecorded. The performances of the predictors with different numbers ofgenes can be assessed based on misclassification error rate,sensitivity, specificity, p values measuring the separation ofKaplan-Meier curves of the two predicted groups.

The Top Scoring Pair (TSP) algorithm first introduced by Geman et al.(2004) can be used. In essence, the algorithm ranks all the gene pairs(genes i and j) based on the absolute difference (Dij) in the frequencyof event where gene i has higher expression value than gene j in samplesamong class C1 to C2. In the cases of there are multiple top scoringpairs (all sharing the same Dij), the top pair by a secondary rank scorethat measures the magnitude to which inversions of gene expressionlevels occur from one class to the other within a pair of genes isselected. The top pair with highest frequency of absolute Dij>2 fold inall samples will be selected as candidate pair. The candidate pair canthen be assessed in an independent testing data set. Leave-one-out crossvalidation (LOOCV) can be carried out in the training data set toevaluate how the algorithm perform. The performances of the predictorscan be assessed based on maximum misclassification error rate. All thestatistical analyses can be done using R (R Development Core Team,2006).

A review of the methods and statistic tools useful for determining areference level can be found in James Westgard, Ph.D., Basic MethodsValidation, 3d edition (2008), which is hereby incorporated by referencein its entirety. Specific references are made to Chapter 9 (“How isreportable range of a method determined”) and Chapter 15 (“How is areference interval verified”).

Clinically reportable range (CRR) is the range of analyte values that amethod can measure, allowing for specimen dilution, concentration, orother pretreatment used to extend the direct analytical measurementrange. As provided in the Basic Methods Validation by Dr. Westgard, theexperiment to be performed is often called a “linearity experiment,”though there technically is no requirement that a method provide alinear response unless two-point calibration is being used. This rangecan also be referred as the “linear range,” “analytical range,” or“working range” for a method.

The reportable range is assessed by inspection of the linearity graph.That inspection can involve manually drawing the best straight linethrough the linear portion of the points, drawing a point-to-point linethrough all the points then comparing with the best straight line, orfitting a regression line through the points in the linear range. Thereare more complicated statistical calculations that are recommended insome guidelines, such as Clinical Laboratory Standards Institute(CLSI)'s EP-6 protocol for evaluating the linearity of analyticalmethods. But it is commonly accepted that the reportable range can beadequately determined from a “visual” assessment, i.e., by manuallydrawing the best straight line that fits the lowest points in theseries. The Clinical Laboratory Standards Institute (CLSI) recommends aminimum of at least 4- preferably 5-different levels of concentrations.More than 5 can be used, particularly if the upper limit of reportablerange needs to be maximized, but 5 levels are convenient and almostalways sufficient.

A reference interval is typically established by assaying specimens thatare obtained from individuals that meet carefully defined criteria(reference sample group). Protocols such as those of the InternationalFederation of Clinical Chemistry (IFCC) Expert Panel on Theory ofReference Values and the CLSI delineate comprehensive systematicprocesses that use carefully selected reference sample groups toestablish reference intervals. These protocols typically need a minimumof 120 reference individuals for each group (or subgroup) that needs tobe characterized.

The CLSI Approved Guideline C28-A2 describes different ways for alaboratory to validate the transference of established referenceintervals to the individual laboratory that includes 1. Divine judgment,wherein the laboratory simply reviews the information submitted andsubjectively verifies that the reference intervals are applicable to theadopting laboratory's patient population and test methods; 2.Verification with 20 samples, wherein experimental validation isperformed by collecting and analyzing specimens from 20 individuals whorepresent the reference sample population; 3. Estimation with 60samples, wherein an experimental validation is performed by collectingand analyzing specimens from 60 individuals who represent the referencesample population, and the actual reference interval is estimated andcompared to the claimed or reported interval using a statistical formulacomparing the means and standard deviations of the two populations; and4. Calculation from comparative method, wherein one can adjust orcorrect the claimed or reported reference intervals on the basis of theobserved methodological bias and the mathematical relationshipdemonstrated between the analytical methods being used.

A person of ordinary skill in the art would understand that thereference expression level of the biomarkers disclosed herein as well asthe reference ratios between two biomarkers can be determined by one ormore methods as provided herein or other methods known in the art.

Accordingly, in some embodiments, the methods provided herein include

a) determining the reference expression level of a biomarker selectedfrom the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM;and

b) administering a therapeutically effective amount of an FTI to acancer patient if

(i) the expression level of KIR2DS2 in a sample from the cancer patientis higher than the reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the cancer patientis lower than the reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the cancerpatient is higher than the reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the cancer patientis lower than the reference expression level of KIR2DL5; or

(v) the expression level of GZMM in a sample from the cancer patient ishigher than the reference expression level of GZMM; or any combinationof (i)-(v).

In some embodiments, the expression level of KIR2DL5 is the totalexpression levels of KIR2DL5A and KIR2DL5B. In some embodiments, theexpression level of KIR2DL5 is the expression level of KIR2DL5A. In someembodiments, the expression level of KIR2DL5 is the expression level ofKIR2DL5B.

Accordingly, in some embodiments, the methods provided herein include

a) determining the reference mRNA level of a biomarker selected from thegroup consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM; and

b) administering a therapeutically effective amount of an FTI to acancer patient if

(i) the mRNA level of KIR2DS2 in a sample from the cancer patient ishigher than the reference mRNA level of KIR2DS2;

(ii) the mRNA level of KIR2DL2 in a sample from the cancer patient islower than the reference mRNA level of KIR2DL2;

(iii) the mRNA level of KIR2DS5 in a sample from the cancer patient ishigher than the reference mRNA level of KIR2DS5;

(iv) the mRNA level of KIR2DL5 in a sample from the cancer patient islower than the reference mRNA level of KIR2DL5; or

(v) the mRNA level of GZMM in a sample from the cancer patient is higherthan the reference mRNA level of GZMM; or any combination of (i)-(v).

In some embodiments, the mRNA level of KIR2DL5 is the total mRNA levelsof KIR2DL5A and KIR2DL5B. In some embodiments, the mRNA level of KIR2DL5is the mRNA level of KIR2DL5A. In some embodiments, the mRNA level ofKIR2DL5 is the mRNA level of KIR2DL5B.

Accordingly, in some embodiments, the methods provided herein include

a) determining the reference protein level of a biomarker selected fromthe group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM; and

b) administering a therapeutically effective amount of an FTI to acancer patient if

(i) the protein level of KIR2DS2 in a sample from the cancer patient ishigher than the reference protein level of KIR2DS2;

(ii) the protein level of KIR2DL2 in a sample from the cancer patient islower than the reference protein level of KIR2DL2;

(iii) the protein level of KIR2DS5 in a sample from the cancer patientis higher than the reference protein level of KIR2DS5;

(iv) the protein level of KIR2DL5 in a sample from the cancer patient islower than the reference protein level of KIR2DL5; or

(v) the protein level of GZMM in a sample from the cancer patient ishigher than the reference protein level of GZMM; or any combination of(i)-(v).

In some embodiments, the protein level of KIR2DL5 is the total proteinlevels of KIR2DL5A and KIR2DL5B. In some embodiments, the protein levelof KIR2DL5 is the protein level of KIR2DL5A. In some embodiments, theprotein level of KIR2DL5 is the protein level of KIR2DL5B.

In some embodiments, the methods provided herein include

a) determining the reference 2DS2/2DL2 ratio, or the reference 2DS5/2DL5ratio; and

b) administering a therapeutically effective amount of an FTI to acancer patient if

(i) the 2DS2/2DL2 ratio in a sample from the cancer patient is higherthan the reference 2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in a sample from the cancer patient is higherthan the reference 2DS5/2DL5 ratio; or both (i) and (ii).

In some embodiments, the 2DS2/2DL2 ratio is the ratio of KIR2DS2 mRNAlevel to KIR2DL2 mRNA level. In some embodiments, the 2DS2/2DL2 ratio isthe ratio of KIR2DS2 protein level to KIR2DL2 protein level. In someembodiments, the 2DS5/2DL5 ratio is the ratio of KIR2DS5 mRNA level toKIR2DL5 mRNA level. In some embodiments, the 2DS5/2DL5 ratio is theratio of KIR2DS5 protein level to KIR2DL5 protein level.

3.5. CANCERS

Provided herein are methods to treat a cancer in a subject with an FTI,and methods for selecting cancer patients for an FTI treatment. Thecancer can be a hematopoietic cancer or a solid tumor. Provided hereinare also methods to treat a premalignant condition in a subject with anFTI, and methods for selecting patients with a premalignant conditionfor an FTI treatment. In some embodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods to treat ahematopoietic cancer in a subject with an FTI or selecting cancerpatients for an FTI treatment. Hematologic cancers are cancers of theblood or bone marrow. Examples of hematological (or hematogenous)cancers include leukemias, lymphoma, and myelodysplastic syndrome (MDS).

Leukemia refers to malignant neoplasms of the blood-forming tissues.Various forms of leukemias are described, for example, in U.S. Pat. No.7,393,862 and U.S. provisional patent application No. 60/380,842, filedMay 17, 2002, the entireties of which are incorporated herein byreference. Although viruses reportedly cause several forms of leukemiain animals, causes of leukemia in humans are to a large extent unknown.The Merck Manual, 944-952 (17^(th) ed. 1999). Transformation tomalignancy typically occurs in a single cell through two or more stepswith subsequent proliferation and clonal expansion. In some leukemias,specific chromosomal translocations have been identified with consistentleukemic cell morphology and special clinical features (e.g.,translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and17 in acute promyelocytic leukemia). Acute leukemias are predominantlyundifferentiated cell populations and chronic leukemias more mature cellforms.

Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. The Merck Manual, 946-949 (17^(th) ed.1999). They may be further subdivided by their morphologic andcytochemical appearance according to the French-American-British (FAB)classification or according to their type and degree of differentiation.The use of specific B- and T-cell and myeloid-antigen monoclonalantibodies are most helpful for classification. ALL is predominantly achildhood disease which is established by laboratory findings and bonemarrow examination. ANLL, also known as acute myelogenous leukemia orAML, occurs at all ages and is the more common acute leukemia amongadults; it is the form usually associated with irradiation as acausative agent. In some embodiments, provided herein are methods fortreating a AML patient with an FTI, or methods for selecting patientsfor FTI treatment.

Standard procedures treat AML patients usually include 2 chemotherapy(chemo) phases: remission induction (or induction) and consolidation(post-remission therapy). The first part of treatment (remissioninduction) is aimed at getting rid of as many leukemia cells aspossible. The intensity of the treatment can depend on a person's ageand health. Intensive chemotherapy is often given to people under theage of 60. Some older patients in good health can benefit from similaror slightly less intensive treatment. People who are much older or arein poor health are not suitable for intensive chemotherapies.

In younger patients, such as those under 60, induction often involvestreatment with 2 chemo drugs, cytarabine (ara-C) and an anthracyclinedrug such as daunorubicin (daunomycin) or idarubicin. Sometimes a thirddrug, cladribine (Leustatin, 2-CdA), is given as well. The chemo isusually given in the hospital and lasts about a week. In rare caseswhere the leukemia has spread to the brain or spinal cord, chemo mayalso be given into the cerebrospinal fluid (CSF). Radiation therapymight be used as well.

Induction is considered successful if remission is achieved. However,the AML in some patients can be refractory to induction. In patients whorespond to induction, further treatment is then given to try to destroyremaining leukemia cells and help prevent a relapse, which is calledconsolidation. For younger patients, the main options for consolidationtherapy are: several cycles of high-dose cytarabine (ara-C) chemo(sometimes known as HiDAC); allogeneic (donor) stem cell transplant; andautologous stem cell transplant.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic(CML). The Merck Manual, 949-952 (17^(th) ed. 1999). CLL ischaracterized by the appearance of mature lymphocytes in blood, bonemarrow, and lymphoid organs. The hallmark of CLL is sustained, absolutelymphocytosis (>5,000/μL) and an increase of lymphocytes in the bonemarrow. Most CLL patients also have clonal expansion of lymphocytes withB-cell characteristics. CLL is a disease of middle or old age. In CML,the characteristic feature is the predominance of granulocytic cells ofall stages of differentiation in blood, bone marrow, liver, spleen, andother organs. In the symptomatic patient at diagnosis, the total whiteblood cell (WBC) count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome. Bone marrow stromal cells are well knownto support CLL disease progression and resistance to chemotherapy.Disrupting the interactions between CLL cells and stromal cells is anadditional target of CLL chemotherapy.

Additionally, other forms of CLL include prolymphocytic leukemia (PLL),Large granular lymphocyte (LGL) leukemia, Hairy cell leukemia (HCL). Thecancer cells in PLL are similar to normal cells calledprolymphocytes—immature forms of B lymphocytes (B-PLL) or T lymphocytes(T-PLL). Both B-PLL and T-PLL tend to be more aggressive than the usualtype of CLL. The cancer cells of LGL are large and have features ofeither T cells or NK cells. Most LGL leukemias are slow-growing, but asmall number are more aggressive. HCL is another cancer of lymphocytesthat tends to progress slowly, and accounts for about 2% of allleukemias. The cancer cells are a type of B lymphocyte but are differentfrom those seen in CLL.

Chronic myelomonocytic leukemia (CMML) is classified as amyelodysplastic/myeloproliferative neoplasm by the 2008 World HealthOrganization classification of hematopoietic tumors. CMML patients havea high number of monocytes in their blood (at least 1,000 per mm³). Twoclasses-myelodysplastic and myeloproliferative-have been distinguishedupon the level of the white blood cell count (threshold 13 G/L). Often,the monocyte count is much higher, causing their total white blood cellcount to become very high as well. Usually there are abnormal cells inthe bone marrow, but the amount of blasts is below 20%. About 15% to 30%of CMML patients go on to develop acute myeloid leukemia. The diagnosisof CMML rests on a combination of morphologic, histopathologic andchromosomal abnormalities in the bone marrow. The Mayo prognostic modelclassified CMML patients into three risk groups based on: increasedabsolute monocyte count, presence of circulating blasts, hemoglobin <10gm/dL and platelets <100×10⁹/L. The median survival was 32 months, 18.5months and 10 months in the low, intermediate, and high-risk groups,respectively. The Groupe Francophone des (GFM) score segregated CMMLpatients into three risk groups based on: age >65 years, WBC >15×10⁹/L,anemia, platelets <100×10⁹/L, and ASXL1 mutation status. After a medianfollow-up of 2.5 years, survival ranged from not reached in the low-riskgroup to 14.4 months in the high-risk group.

Lymphoma refers to cancers that originate in the lymphatic system.Lymphoma is characterized by malignant neoplasms of lymphocytes—Blymphocytes (B cell lymphoma), T lymphocytes (T-cell lymphoma), andnatural killer cells (NK cell lymphoma). Lymphoma generally starts inlymph nodes or collections of lymphatic tissue in organs including, butnot limited to, the stomach or intestines. Lymphoma may involve themarrow and the blood in some cases. Lymphoma may spread from one site toother parts of the body.

The treatments of various forms of lymphomas are described, for example,in U.S. Pat. No. 7,468,363, the entirety of which is incorporated hereinby reference. Such lymphomas include, but are not limited to, Hodgkin'slymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activatedB-cell lymphoma, Diffuse Large B-Cell Lymphoma (DLBCL), mantle celllymphoma (MCL), follicular lymphoma (FL; including but not limited to FLgrade I, FL grade II), follicular center lymphoma, transformed lymphoma,lymphocytic lymphoma of intermediate differentiation, intermediatelymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocyticlymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved celllymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Celllymphoma (CTCL) and mantle zone lymphoma and low grade follicularlymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common cancer for bothmen and women in the United States, with an estimated 63,190 new casesand 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007;57(1):43-66. The probability of developing NHL increases with age andthe incidence of NHL in the elderly has been steadily increasing in thepast decade, causing concern with the aging trend of the U.S.population. Id. Clarke C A, et al., Cancer 2002; 94(7):2015-2023.

DLBCL accounts for approximately one-third of non-Hodgkin's lymphomas.While some DLBCL patients are cured with traditional chemotherapy, theremainders die from the disease. Anticancer drugs cause rapid andpersistent depletion of lymphocytes, possibly by direct apoptosisinduction in mature T and B cells. See K. Stahnke. et al., Blood 2001,98:3066-3073. Absolute lymphocyte count (ALC) has been shown to be aprognostic factor in follicular non-Hodgkin's lymphoma and recentresults have suggested that ALC at diagnosis is an important prognosticfactor in DLBCL.

DLBCL can be divided into distinct molecular subtypes according to theirgene profiling patterns: germinal-center B-cell-like DLBCL (GCB-DLBCL),activated B-cell-like DLBCL (ABC-DLBCL), and primary mediastinal B-celllymphoma (PMBL) or unclassified type. These subtypes are characterizedby distinct differences in survival, chemo-responsiveness, and signalingpathway dependence, particularly the NF-κB pathway. See D. Kim et al.,Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings PartI. Vol 25, No. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al.,Blood 2005; 106: 3183-90; Ngo V. N. et al., Nature 2011; 470: 115-9.Such differences have prompted the search for more effective andsubtype-specific treatment strategies in DLBCL. In addition to the acuteand chronic categorization, neoplasms are also categorized based uponthe cells giving rise to such disorder into precursor or peripheral. Seee.g., U.S. patent Publication No. 2008/0051379, the disclosure of whichis incorporated herein by reference in its entirety. Precursor neoplasmsinclude ALLs and lymphoblastic lymphomas and occur in lymphocytes beforethey have differentiated into either a T- or B-cell. Peripheralneoplasms are those that occur in lymphocytes that have differentiatedinto either T- or B-cells. Such peripheral neoplasms include, but arenot limited to, B-cell CLL, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma,extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoidtissue, nodal marginal zone lymphoma, splenic marginal zone lymphoma,hairy cell leukemia, plasmacytoma, Diffuse large B-cell lymphoma (DLBCL)and Burkitt lymphoma. In over 95 percent of CLL cases, the clonalexpansion is of a B cell lineage. See Cancer: Principles & Practice ofOncology (3rd Edition) (1989) (pp. 1843-1847). In less than 5 percent ofCLL cases, the tumor cells have a T-cell phenotype. Notwithstandingthese classifications, however, the pathological impairment of normalhematopoiesis is the hallmark of all leukemias.

PTCL consists of a group of rare and usually aggressive (fast-growing)NHLs that develop from mature T-cells. PTCLs collectively account forabout 4 to 10 percent of all NHL cases, corresponding to an annualincidence of 2,800-7,200 patients per year in the United States. By someestimates, the incidence of PTCL is growing significantly, and theincreasing incidence may be driven by an aging population. PTCLs aresub-classified into various subtypes, each of which are typicallyconsidered to be separate diseases based on their distinct clinicaldifferences. Most of these subtypes are rare; the three most commonsubtypes of PTCL not otherwise specified, anaplastic large-celllymphoma, or ALCL, and angioimmunoblastic T-cell lymphoma, thatcollectively account for approximately 70 percent of all PTCLs in theUnited States. ALCL can be cutaneous ALCL or systemic ALCL

For most PTCL subtypes, the frontline treatment regimen is typicallycombination chemotherapy, such as CHOP (cyclophosphamide, doxorubicin,vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin,cyclophosphamide, prednisone), or other multi-drug regimens. Patientswho relapse or are refractory to frontline treatments are typicallytreated with gemcitabine in combination with other chemotherapies,including vinorelbine (Navelbine®) and doxorubicin (Doxil®) in a regimencalled GND, or other chemotherapy regimens such as DHAP (dexamethasone,cytarabine, cisplatin) or ESHAP (etoposide, methylprednisolone,cytarabine, and cisplatin).

Because most patients with PTCL will relapse, some oncologists recommendgiving high-dose chemotherapy followed by an autologous stem celltransplant to some patients who had a good response to their initialchemotherapy. Recent, non-cytotoxic therapies that have been approvedfor relapsed or refractory PTCL, such as pralatrexate (Folotyn®),romidepsin (Istodax®) and belinostat (Beleodaq®), are associated withrelatively low objective response rates (25-27% overall response rate,or ORR) and relatively short durations of response (8.2-9.4 months).Accordingly, the treatment of relapsed/refractory PTCL remains asignificant unmet medical need.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow.Normally, plasma cells produce antibodies and play a key role in immunefunction. However, uncontrolled growth of these cells leads to bone painand fractures, anemia, infections, and other complications. Multiplemyeloma is the second most common hematological malignancy, although theexact causes of multiple myeloma remain unknown. Multiple myeloma causeshigh levels of proteins in the blood, urine, and organs, including butnot limited to M-protein and other immunoglobulins (antibodies),albumin, and beta-2-microglobulin. M-protein, short for monoclonalprotein, also known as paraprotein, is a particularly abnormal proteinproduced by the myeloma plasma cells and can be found in the blood orurine of almost all patients with multiple myeloma.

Skeletal symptoms, including bone pain, are among the most clinicallysignificant symptoms of multiple myeloma. Malignant plasma cells releaseosteoclast stimulating factors (including IL-1, IL-6 and TNF) whichcause calcium to be leached from bones causing lytic lesions;hypercalcemia is another symptom. The osteoclast stimulating factors,also referred to as cytokines, may prevent apoptosis, or death ofmyeloma cells. Fifty percent of patients have radiologically detectablemyeloma-related skeletal lesions at diagnosis. Other common clinicalsymptoms for multiple myeloma include polyneuropathy, anemia,hyperviscosity, infections, and renal insufficiency.

Bone marrow stromal cells are well known to support multiple myelomadisease progression and resistance to chemotherapy. Disrupting theinteractions between multiple myeloma cells and stromal cells is anadditional target of multiple myeloma chemotherapy.

Myelodysplastic syndrome (MDS) refers to a diverse group ofhematopoietic stem cell disorders. MDS can be characterized by acellular marrow with impaired morphology and maturation(dysmyelopoiesis), ineffective blood cell production, or hematopoiesis,leading to low blood cell counts, or cytopenias, and high risk ofprogression to acute myeloid leukemia, resulting from ineffective bloodcell production. See The Merck Manual 953 (17th ed. 1999) and List etal., 1990, J Clin. Oncol. 8:1424.

As a group of hematopoietic stem cell malignancies with significantmorbidity and mortality, MDS is a highly heterogeneous disease, and theseverity of symptoms and disease progression can vary widely amongpatients. The current standard clinical tool to evaluate riskstratification and treatment options is the revised InternationalPrognostic Scoring System, or IPSS-R. The IPSS-R differentiates patientsinto five risk groups (Very Low, Low, Intermediate, High, Very High)based on evaluation of cytogenetics, percentage of blasts(undifferentiated blood cells) in the bone marrow, hemoglobin levels,and platelet and neutrophil counts. The WHO also suggested stratifyingMDS patients by a del (5q) abnormality.

According to the ACS, the annual incidence of MDS is approximately13,000 patients in the United States, the majority of which are 60 yearsof age or older. The estimated prevalence is over 60,000 patients in theUnited States. Approximately 75% of patients fall into the IPSS-R riskcategories of Very Low, Low, and Intermediate, or collectively known aslower risk MDS.

The initial hematopoietic stem cell injury can be from causes such as,but not limited to, cytotoxic chemotherapy, radiation, virus, chemicalexposure, and genetic predisposition. A clonal mutation predominatesover bone marrow, suppressing healthy stem cells. In the early stages ofMDS, the main cause of cytopenias is increased programmed cell death(apoptosis). As the disease progresses and converts into leukemia, genemutation rarely occurs and a proliferation of leukemic cells overwhelmsthe healthy marrow. The disease course differs, with some cases behavingas an indolent disease and others behaving aggressively with a veryshort clinical course that converts into an acute form of leukemia.

An international group of hematologists, the French-American-British(FAB) Cooperative Group, classified MDS disorders into five subgroups,differentiating them from AML. The Merck Manual 954 (17^(th) ed. 1999);Bennett J. M., et al., Ann. Intern. Med. 1985 October, 103(4): 620-5;and Besa E. C., Med. Clin. North Am. 1992 May, 76(3): 599-617. Anunderlying trilineage dysplastic change in the bone marrow cells of thepatients is found in all subtypes.

There are two subgroups of refractory anemia characterized by fivepercent or less myeloblasts in bone marrow: (1) refractory anemia (RA)and; (2) RA with ringed sideroblasts (RARS), defined morphologically ashaving 15% erythroid cells with abnormal ringed sideroblasts, reflectingan abnormal iron accumulation in the mitochondria. Both have a prolongedclinical course and low incidence of progression to acute leukemia. BesaE. C., Med. Clin. North Am. 1992 May, 76(3): 599-617.

There are two subgroups of refractory anemias with greater than fivepercent mycloblasts: (1) RA with excess blasts (RAEB), defined as 6-20%myeloblasts, and (2) RAEB in transformation (RAEB-T), with 21-30%myeloblasts. The higher the percentage of myeloblasts, the shorter theclinical course and the closer the disease is to acute myelogenousleukemia. Patient transition from early to more advanced stagesindicates that these subtypes are merely stages of disease rather thandistinct entities. Elderly patients with MDS with trilineage dysplasiaand greater than 30% myeloblasts who progress to acute leukemia areoften considered to have a poor prognosis because their response rate tochemotherapy is lower than de novo acute myeloid leukemia patients. Thefifth type of MDS, the most difficult to classify, is CMML. This subtypecan have any percentage of myeloblasts but presents with a monocytosisof 1000/dL or more. It may be associated with splenomegaly. This subtypeoverlaps with a myeloproliferative disorder and may have an intermediateclinical course. It is differentiated from the classic CML that ischaracterized by a negative Ph chromosome.

MDS is primarily a disease of elderly people, with the median onset inthe seventh decade of life. The median age of these patients is 65years, with ages ranging from the early third decade of life to as oldas 80 years or older. The syndrome may occur in any age group, includingthe pediatric population. Patients who survive malignancy treatment withalkylating agents, with or without radiotherapy, have a high incidenceof developing MDS or secondary acute leukemia. About 60-70% of patientsdo not have an obvious exposure or cause for MDS, and are classified asprimary MDS patients.

The treatment of MDS is based on the stage and the mechanism of thedisease that predominates the particular phase of the disease process.Bone marrow transplantation has been used in patients with poorprognosis or late-stage MDS. Epstein and Slease, 1985, Surg. Ann.17:125. An alternative approach to therapy for MDS is the use ofhematopoietic growth factors or cytokines to stimulate blood celldevelopment in a recipient. Dexter, 1987, J. Cell Sci. 88:1; Moore,1991, Annu. Rev. Immunol. 9:159; and Besa E. C., Med. Clin. North Am.1992 May, 76(3): 599-617. The treatment of MDS using immunomodulatorycompounds is described in U.S. Pat. No. 7,189,740, the entirety of whichis hereby incorporated by reference.

Therapeutic options fall into three categories including supportivecare, low intensity and high intensity therapy. Supportive care includesthe use red blood cell and platelet transfusions and hematopoieticcytokines such as erythropoiesis stimulating agents or colonystimulating factors to improve blood counts. Low intensity therapiesinclude hypomethylating agents such as azacytidine (Vidaza®) anddecitabine (Dacogen®), biological response modifiers such aslenalidomide (Revlimid®), and immunosuppressive treatments such ascyclosporine A or antithymocyte globulin. High intensity therapiesinclude chemotherapeutic agents such as idarubicin, azacytidine,fludarabine and topotecan, and hematopoietic stem cell transplants, orHSCT.

National Comprehensive Cancer Network, or NCCN, guidelines recommendthat lower risk patients (IPSS-R groups Very Low, Low, Intermediate)receive supportive care or low intensity therapies with the majortherapeutic goal of hematologic improvement, or HI. NCCN guidelinesrecommend that higher risk patients (IPSS-R groups High, Very High)receive more aggressive treatment with high intensity therapies. In somecases, high risk patients are unable to tolerate chemotherapy, and mayelect lower intensity regimens. Despite currently available treatments,a substantial portion of MDS patients lack effective therapies and NCCNguidelines recommend clinical trials as additional therapeutic options.Treatment of MDS remains a significant unmet need requiring thedevelopment of novel therapies.

Accordingly, in some embodiments, provided herein are methods to treathematopoietic cancer in a subject with FTI, or selecting a hematopoieticcancer patient for an FTI treatment, wherein the hematopoietic cancerpatient is a carrier of KIR2DS2 or a carrier of KIR2DS5, or both; orwherein

(i) the expression level of KIR2DS2 in a sample from the hematopoieticcancer patient is higher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the hematopoieticcancer patient is lower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the hematopoieticcancer patient is higher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the hematopoieticcancer patient is lower than a reference expression level of KIR2DL5;

(v) the expression level of GZMM in a sample from the hematopoieticcancer patient is higher than a reference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in a sample from the hematopoietic cancer patient ishigher than a reference ratio of an expression level of KIR2DS2 to anexpression level of KIR2DL2; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in a sample from the hematopoietic cancer patient ishigher than a reference ratio of an expression level of KIR2DS5 to anexpression level of KIR2DL5; or any combination of (i)-(vii).

In some embodiments, provided herein are methods to treat ahematopoietic cancer in a subject with FTI, or selecting a hematopoieticcancer patient for an FTI treatment. Hematological cancers includeleukemias, including acute leukemias (such as acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblasts, promyeiocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, chronic myeloicleukemia, and chronic lymphocytic leukemia), chronic myelomonocyticleukemia, juvenile myelomonocytic leukemia, polycythemia vera, NK cellleukemia, lymphoma, NK cell lymphoma, Hodgkin's disease, non-Hodgkin'slymphoma (indolent and high grade forms), multiple myeloma, peripheralT-cell lymphomas, cutaneous T-Cell lymphoma, Waldenstrom'smacroglobulinemia, heavy chain disease, myeiodysplastic syndrome,agnogenic myeloid metaplasia, familial erythrophagocyticlymphohistiocytosis, hairy cell leukemia and myelodysplasia.

In some embodiments, the hematopoietic cancer to be treated by methodsprovided herein can be AML, MDS, CMML, NK cell lymphoma, NK cellleukemia, CTCL, PTCL, CML. In some embodiments, the hematopoietic canceris AML. In some embodiments, the hematopoietic cancer is MDS. In someembodiments, the MDS is lower risk MDS. In some embodiments, thehematopoietic cancer is CMML. The CMML can be low risk CMML,intermediate risk CMML, or high risk CMML. The CMML can bemyelodysplastic CMML or myeloproliferative CMML. In some embodiments,the CMML is NRAS/KRAS wild type CMML. In some embodiments, thehematopoietic cancer is NK lymphoma. In some embodiments, thehematopoietic cancer is NK leukemia. In some embodiments, thehematopoietic cancer is CTCL. In some embodiments, the hematopoieticcancer is PTCL. In some embodiments, the PTCL is refractory or relapsedPTCL.

In some embodiments, provided herein are methods to treat MDS in asubject with FTI, or selecting a MDS patient for an FTI treatment,wherein the MDS patient is a carrier of KIR2DS2, KIR2DS5, or HLA-C2, orany combination thereof; or wherein

(i) the expression level of KIR2DS2 in a sample from the MDS patient ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the MDS patient islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the MDS patientis higher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the MDS patient islower than a reference expression level of KIR2DL5;

(v) the expression level of GZMM in a sample from the MDS patient ishigher than a reference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in a sample from the MDS patient is higher than areference ratio of an expression level of KIR2DS2 to an expression levelof KIR2DL2; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in a sample from the MDS patient is higher than areference ratio of an expression level of KIR2DS5 to an expression levelof KIR2DL5; or any combination thereof.

In some embodiments, provided herein are methods to treat a lower riskMDS in a subject with FTI, or selecting a lower risk MDS patient for anFTI treatment, wherein the lower risk MDS patient is a carrier ofKIR2DS2, KIR2DS5, or HLA-C2, or any combination thereof; or wherein

(i) the expression level of KIR2DS2 in a sample from the MDS patient ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the MDS patient islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the MDS patientis higher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the MDS patient islower than a reference expression level of KIR2DL5;

(v) the expression level of GZMM in a sample from the MDS patient ishigher than a reference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in a sample from the lower risk MDS patient is higherthan a reference ratio of an expression level of KIR2DS2 to anexpression level of KIR2DL2; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in a sample from the lower risk MDS patient is higherthan a reference ratio of an expression level of KIR2DS5 to anexpression level of KIR2DL5; or any combination thereof.

In some embodiments, provided herein are methods to treat AML in asubject with FTI, or selecting a AML patient for an FTI treatment,wherein the AML patient is a carrier of KIR2DS2, KIR2DS5, or HLA-C2, orany combination thereof; or wherein

(i) the expression level of KIR2DS2 in a sample from the AML patient ishigher than a reference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in a sample from the AML patient islower than a reference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in a sample from the AML patientis higher than a reference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from the AML patient islower than a reference expression level of KIR2DL5;

(v) the expression level of GZMM in a sample from the AML patient ishigher than a reference expression level of GZMM;

(vi) the ratio of the expression level of KIR2DS2 to the expressionlevel of KIR2DL2 in a sample from the AML patient is higher than areference ratio of an expression level of KIR2DS2 to an expression levelof KIR2DL2; or

(vii) the ratio of the expression level of KIR2DS5 to the expressionlevel of KIR2DL5 in a sample from the AML patient is higher than areference ratio of an expression level of KIR2DS5 to an expression levelof KIR2DL5; or any combination thereof.

In some embodiments, the AML patient is post-remission induction. Insome embodiments, the AML patient post-transplantation. In someembodiments, the AML patient is over age 60 or otherwise unfit forremission induction. In some embodiments, the AML patient is over age65, 70, or 75. In some embodiments, the AML patient is refractory tostandard chemotherapy. In some embodiments, the AML patient is arelapsed patient.

In some embodiments, provided herein are methods to treat a solid tumor.Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). The solid tumorto be treated with the methods of the invention can be sarcomas andcarcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, pheochromocytomas sebaceous gland carcinoma, papillarycarcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma(such as brainstem glioma and mixed gliomas), glioblastoma (also knownas glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,meduloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

In some embodiments, provided herein are methods to treat a solid tumor,wherein the solid tumor is malignant melanoma, adrenal carcinoma, breastcarcinoma, renal cell cancer, carcinoma of the pancreas, non-small-celllung carcinoma (NSCLC) or carcinoma of unknown primary. Drugs commonlyadministered to patients with various types or stages of solid tumorsinclude, but are not limited to, celebrex, etoposide, cyclophosphamide,docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combinationthereof.

In some embodiments, the solid tumor to be treated by methods providedherein can be thyroid cancer, head and neck cancers, urothelial cancers,salivary cancers, cancers of the upper digestive tract, bladder cancer,breast cancer, ovarian cancer, brain cancer, gastric cancer, prostatecancer, lung cancer, colon cancer, skin cancer, liver cancer, andpancreatic cancer. In some embodiments, the bladder cancer to be treatedby methods provided herein can be transitional cell carcinoma.

In some embodiments, the solid tumor to be treated by methods providedherein can be selected from the groups consisting of carcinoma,melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the premalignant conditions to be treated bymethods provided herein can be actinic cheilitis, Barrett's esophagus,atrophic gastritis, ductal carcinoma in situ, Dyskeratosis congenita,Sideropenic dysphagia, Lichen planus, Oral submucous fibrosis, Solarelastosis, cervical dysplasia, polyps, leukoplakia, erythroplakia,squamous intraepithelial lesion, a pre-malignant disorder, or apre-malignant immunoproliferative disorder.

3.6. EXEMPLARY FTIS AND DOSAGES

In some embodiments, the methods for treating cancer in a subjectinclude KIR typing the subject, and administering a therapeuticallyeffective amount of tipifarnib to the subject, wherein the subject is acarrier of KIR2DS2 or KIR2DS5, or a carrier of both KIR2DS2 and KIR2DS5.In some embodiments, the subject is also a carrier of HLA-C2. In someembodiments, the methods include administering the subject with anotherFTI described herein or otherwise known in the art. In some embodiments,the FTI is selected from the group consisting of tipifarnib, arglabin,perrilyl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277,L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the method for treating cancer in a subjectincludes determining expression level of a biomarker in a sample fromthe subject, wherein the biomarker is selected from the group consistingof KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, and administering atherapeutically effective amount of tipifarnib to the subject wherein

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5; or

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM; or any combination of (i)-(v). Insome embodiments, the methods include administering the subject withanother FTI described herein or otherwise known in the art. In someembodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

Additionally, the method of treating cancer in a subject includesdetermining expression levels of KIR2DS2 and KIR2DL2, or KIR2DS5 andKIR2DL5 in a sample from the subject, and administering atherapeutically effective amount of tipifarnib to the subject, wherein

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; or both (i) and (ii). In some embodiments, the methodsinclude administering the subject with another FTI described herein orotherwise known in the art. In some embodiments, the FTI is selectedfrom the group consisting of tipifarnib, arglabin, perrilyl alcohol,lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754,R208176, AZD3409, and BMS-214662.

In some embodiments, the method for treating a hematological cancer in asubject include KIR typing the subject, and administering atherapeutically effective amount of tipifarnib to the subject, whereinthe subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of bothKIR2DS2 and KIR2DS5. In some embodiments, the subject is also a carrierof HLA-C2. In some embodiments, the methods include administering thesubject with another FTI described herein or otherwise known in the art.In some embodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the method for treating a hematological cancer in asubject includes determining expression level of a biomarker in a samplefrom the subject, wherein the biomarker is selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, andadministering a therapeutically effective amount of tipifarnib to thesubject wherein

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5; or

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM; or any combination of (i)-(v). Insome embodiments, the methods include administering the subject withanother FTI described herein or otherwise known in the art. In someembodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the method of treating a hematological cancer in asubject includes determining expression levels of KIR2DS2 and KIR2DL2,or KIR2DS5 and KIR2DL5 in a sample from the subject, and administering atherapeutically effective amount of tipifarnib to the subject, wherein

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; or both (i) and (ii). In some embodiments, the methodsinclude administering the subject with another FTI described herein orotherwise known in the art. In some embodiments, the FTI is selectedfrom the group consisting of tipifarnib, arglabin, perrilyl alcohol,lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754,R208176, AZD3409, and BMS-214662.

In some embodiments, the method for treating a lower risk MDS in asubject include KIR typing the subject, and administering atherapeutically effective amount of tipifarnib to the subject, whereinthe subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of bothKIR2DS2 and KIR2DS5. In some embodiments, the subject is also a carrierof HLA-C2. In some embodiments, the methods include administering thesubject with another FTI described herein or otherwise known in the art.In some embodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the method for treating a lower risk MDS in asubject includes determining expression level of a biomarker in a samplefrom the subject, wherein the biomarker is selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, andadministering a therapeutically effective amount of tipifarnib to thesubject wherein

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5; or

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM; or any combination of (i)-(v). Insome embodiments, the methods include administering the subject withanother FTI described herein or otherwise known in the art. In someembodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

Additionally, the method of treating a lower risk MDS in a subjectincludes determining expression levels of KIR2DS2 and KIR2DL2, orKIR2DS5 and KIR2DL5 in a sample from the subject, and administering atherapeutically effective amount of tipifarnib to the subject, wherein

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; or both (i) and (ii). In some embodiments, the methodsinclude administering the subject with another FTI described herein orotherwise known in the art. In some embodiments, the FTI is selectedfrom the group consisting of tipifarnib, arglabin, perrilyl alcohol,lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754,R208176, AZD3409, and BMS-214662.

In some embodiments, the FTI is administered orally, parenterally,rectally, or topically. In some embodiments, the FTI is administeredorally.

In some embodiments, tipifarnib is administered orally, parenterally,rectally, or topically. In some embodiments, tipifarnib is administeredorally.

In some embodiments, the FTI is administered at a dose of 1-1000 mg/kgbody weight. In some embodiments, the FTI is administered twice a day.In some embodiments, the FTI is administered at a dose of 200-1200 mgtwice a day. In some embodiments, the FTI is administered at a dose of600 mg twice a day. In some embodiments, the FTI is administered at adose of 900 mg twice a day.

In some embodiments, tipifarnib is administered at a dose of 1-1000mg/kg body weight. In some embodiments, tipifarnib is administered twicea day. In some embodiments, tipifarnib is administered at a dose of200-1200 mg twice a day. In some embodiments, tipifarnib is administeredat a dose of 600 mg twice a day. In some embodiments, tipifarnib isadministered at a dose of 900 mg twice a day.

In some embodiments, the FTI is administered in treatment cycles. Insome embodiments, the FTI is administered in alternative weeks. In someembodiments, the FTI is administered on days 1-7 and 15-21 of a 28-daytreatment cycle. In some embodiments, the FTI is administered orally ata dose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle.

In some embodiments, tipifarnib is administered in treatment cycles. Insome embodiments, tipifarnib is administered in alternative weeks. Insome embodiments, tipifarnib is administered on days 1-7 and 15-21 of a28-day treatment cycle. In some embodiments, tipifarnib is administeredorally at a dose of 900 mg twice a day on days 1-7 and 15-21 of a 28-daytreatment cycle.

In some embodiments, the FTI is administered for at least 3 cycles. Insome embodiments, the FTI is administered for at least 6 cycles. In someembodiments, the FTI is administered for up to 12 cycles. In someembodiments, the FTI is administered orally at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle for at least threecycles.

In some embodiments, tipifarnib is administered for at least 3 cycles.In some embodiments, tipifarnib is administered for at least 6 cycles.In some embodiments, tipifarnib is administered for up to 12 cycles. Insome embodiments, tipifarnib is administered orally at a dose of 900 mgtwice a day on days 1-7 and 15-21 of a 28-day treatment cycle for atleast three cycles.

In some embodiments, the method for treating a lower risk MDS in asubject include KIR typing the subject, and administering tipifarnib tothe subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, ora carrier of both KIR2DS2 and KIR2DS5, and wherein th tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle. In some embodiments, the subject isalso a carrier of HLA-C2.

In some embodiments, the method for treating a lower risk MDS in asubject includes determining expression level of a biomarker in a samplefrom the subject, wherein the biomarker is selected from the groupconsisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, andadministering tipifarnib to the subject wherein

(i) the expression level of KIR2DS2 in the sample is higher than areference expression level of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than areference expression level of KIR2DL2;

(iii) the expression level of KIR2DS5 in the sample is higher than areference expression level of KIR2DS5;

(iv) the expression level of KIR2DL5 in the sample is lower than areference expression level of KIR2DL5; or

(v) the expression level of GZMM in the sample is higher than areference expression level of GZMM; or any combination of (i)-(v); andwherein tipifarnib is administered orally at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle.

Additionally, the method of treating a lower risk MDS in a subjectincludes determining expression levels of KIR2DS2 and KIR2DL2, orKIR2DS5 and KIR2DL5 in a sample from the subject, and administeringtipifarnib to the subject, wherein

(i) the 2DS2/2DL2 ratio in the sample is higher than a reference2DS2/2DL2 ratio; or

(ii) the 2DS5/2DL5 ratio in the sample is higher than a reference2DS5/2DL5 ratio; or both (i) and (ii); and wherein tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle.

3.7. KITS

In certain embodiments, provided herein is a kit for KIR typing asubject. In some embodiments, the kit includes one or more probes thatbind specifically to the genomic DNA, cDNA, or mRNA of the one or moreKIR genes. The KIR genes can include KIR2DS2, KIR2DL2, KIR2DS5, KIRDL5,or any combination thereof. In some embodiments, the kits can furtherinclude an agent for HLA typing. The agent for HLA typing can be one ormore probes that bind specifically to the genomic DNA, cDNA, or mRNA ofthe one or more HLA genes. The HLA genes can include HLA-C1, HLA-C2, orboth.

In certain embodiments, the kit further includes a washing solution. Incertain embodiments, the kit further comprises reagents for genomic DNAisolation or purification means, detection means, as well as positiveand negative controls. In certain embodiments, the kit further includesan instruction for using the kit. In some embodiments, the kit furtherincludes an FTI or a pharmacological composition having an FTI. The kitcan be tailored for in-home use, clinical use, or research use.

In certain embodiments, provided herein is a kit for detecting the mRNAlevel of one or more biomarkers. The one or more biomarker are selectedfrom the group consisting of can include KIR2DS2, KIR2DL2, KIR2DS5,KIRDL5, and GZMM. In certain embodiments, the kit includes one or moreprobes that bind specifically to the mRNAs of the one or morebiomarkers. In certain embodiments, the kit further includes a washingsolution. In certain embodiments, the kit further includes reagents forperforming a hybridization assay, mRNA isolation or purification means,detection means, as well as positive and negative controls. In certainembodiments, the kit further includes an instruction for using the kit.In some embodiments, the kit further includes an FTI or apharmacological composition having an FTI. The kit can be tailored forin-home use, clinical use, or research use.

In certain embodiments, provided herein is a kit for detecting theprotein level of one or more biomarkers. The one or more biomarker areselected from the group consisting of can include KIR2DS2, KIR2DL2,KIR2DS5, KIRDL5, and GZMM. In certain embodiments, the kits includes adipstick coated with an antibody that recognizes the protein biomarker,washing solutions, reagents for performing the assay, protein isolationor purification means, detection means, as well as positive and negativecontrols. In certain embodiments, the kit further includes aninstruction for using the kit. In some embodiments, the kit furtherincludes an FTI or a pharmacological composition having an FTI. The kitcan be tailored for in-home use, clinical use, or research use.

The kits provided herein can employ, for example, a dipstick, amembrane, a chip, a disk, a test strip, a filter, a microsphere, aslide, a multiwell plate, or an optical fiber. The solid support of thekit can be, for example, a plastic, silicon, a metal, a resin, glass, amembrane, a particle, a precipitate, a gel, a polymer, a sheet, asphere, a polysaccharide, a capillary, a film, a plate, or a slide. Thesample can be, for example, a blood sample, a bone marrow sample, a cellculture, a cell line, a tissue, an oral issue, gastrointestinal tissue,an organ, an organelle, a biological fluid, a urine sample, or a skinsample. The biological sample can be, for example, a lymph node biopsy,a bone marrow biopsy, or a sample of peripheral blood tumor cells.

In some embodiments, the kits provided herein include one or morecontainers and components for conducting RT-PCR, qPCR, deep sequencing,NGS, or a microarray. In certain embodiments, the kits provided hereinemploy means for detecting the expression of a biomarker by flowcytometry or immunofluorescence. In other embodiments, the expression ofthe biomarker is measured by ELISA-based methodologies or other similarmethods known in the art.

In certain embodiments, the kits provided herein include components forisolating protein. In another specific embodiment, the pharmaceutical orassay kit includes, in a container, an FTI or a pharmaceuticalcomposition having an FTI, and further includes, in one or morecontainers, components for conducting flow cytometry or an ELISA.

In some embodiments, provided herein are kits for measuring biomarkersproviding the materials necessary to measure the presence of certaingenes, or abundance of one or more of the gene products of the genes ora subset of genes (e.g., one, two, three, four, five or more genes) ofthe biomarkers provided herein. Such kits can include materials andreagents required for measuring DNA, RNA or protein. In someembodiments, such kits include microarrays, wherein the microarray iscomprised of oligonucleotides and/or DNA and/or RNA fragments whichhybridize to one or more of the DNA or mRNA transcripts of one or moreof the genes or a subset of genes of the biomarkers provided herein, orany combination thereof. In some embodiments, such kits can includeprimers for PCR of either the DNA, RNA product or the cDNA copy of theRNA product of the genes or subset of genes. In some embodiments, suchkits can include primers for PCR as well as probes for Quantitative PCR.In some embodiments, such kits can include multiple primers and multipleprobes wherein some of the probes have different fluorophores so as topermit multiplexing of multiple products of a gene product or multiplegene products. In some embodiments, such kits can further includematerials and reagents for synthesizing cDNA from RNA isolated from asample. In some embodiments, such kits can include antibodies specificfor the protein products of a gene or subset of genes of the biomarkersprovided herein. Such kits can additionally include materials andreagents for isolating RNA and/or proteins from a biological sample. Insome embodiments, such kits can include, a computer program productembedded on computer readable media for predicting whether a patient isclinically sensitive to an FTI. In some embodiments, the kits caninclude a computer program product embedded on a computer readable mediaalong with instructions.

In some embodiments, kits for measuring the expression of one or morenucleic acid sequences of a gene or a subset of genes of the biomarkersprovided herein. In a specific embodiment, such kits measure theexpression of one or more nucleic acid sequences associated with a geneor a subset of genes of the biomarkers provided herein. In accordancewith this embodiment, the kits may comprise materials and reagents thatare necessary for measuring the expression of particular nucleic acidsequence products of genes or a subset of genes of the biomarkersprovided herein. For example, a microarray or RT-PCR kit may be producedfor a specific condition and contain only those reagents and materialsnecessary for measuring the levels of specific RNA transcript productsof the genes or a subset of genes of the biomarkers provided herein topredict whether a hematological cancer in a patient is clinicallysensitive to a compound. Alternatively, in some embodiments, the kitscan comprise materials and reagents that are not limited to thoserequired to measure the expression of particular nucleic acid sequencesof any particular gene of the biomarkers provided herein. For example,in certain embodiments, the kits comprise materials and reagentsnecessary for measuring the levels of expression of 1, 2, 3, 4, or 5 ofthe biomarkers provided herein, in addition to reagents and materialsnecessary for measuring the levels of the expression of at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 15, at least 20, at least 25,at least 30, at least 35, at least 40, at least 45, at least 50 or moregenes other than those of the biomarkers provided herein. In otherembodiments, the kits contain reagents and materials necessary formeasuring the levels of expression of at least 1, at least 2, at least3, at least 4, at least 5, or more of the genes of the biomarkersprovided herein, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250,300, 350, 400, 450, or more genes that are genes not of the biomarkersprovided herein, or 1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500,1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150,100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes that aregenes not of the biomarkers provided herein.

For nucleic acid microarray kits, the kits generally include probesattached to a solid support surface. In one such embodiment, probes canbe either be oligonucleotides or longer length probes including probesranging from 150 nucleotides in length to 800 nucleotides in length. Theprobes can be attached to a detectable label. In a specific embodiment,the probes are specific for one or more of the gene products of thebiomarkers provided herein. The microarray kits can include instructionsfor performing the assay and methods for interpreting and analyzing thedata resulting from the performance of the assay. In a specificembodiment, the kits include instructions for predicting whether ahematological cancer in a patient is clinically sensitive to an FTI. Thekits can also include hybridization reagents and/or reagents necessaryfor detecting a signal produced when a probe hybridizes to a targetnucleic acid sequence. Generally, the materials and reagents for themicroarray kits are in one or more containers. Each component of the kitis generally in its own a suitable container.

In certain embodiments, a nucleic acid microarray kit includes materialsand reagents necessary for measuring the levels of expression of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of thegenes identified of the biomarkers provided herein, or a combinationthereof, in addition to reagents and materials necessary for measuringthe levels of the expression of at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 15, at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50 or more genes other than thoseof the biomarkers provided herein. In other embodiments, a nucleic acidmicroarray kit contains reagents and materials necessary for measuringthe levels of expression of at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 15, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50 or more of the genes of thebiomarkers provided herein, or any combination thereof, and 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or more genesthat are not of the biomarkers provided herein, or 1-10, 1-100, 1-150,1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400,25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000or 500-1000 genes that are not of the biomarkers provided herein.

For Quantitative PCR, the kits can include pre-selected primers specificfor particular nucleic acid sequences. The Quantitative PCR kits canalso include enzymes suitable for amplifying nucleic acids (e.g.,polymerases such as Taq), and deoxynucleotides and buffers needed forthe reaction mixture for amplification. The Quantitative PCR kits canalso include probes specific for the nucleic acid sequences associatedwith or indicative of a condition. The probes can be labeled with afluorophore. The probes can also be labeled with a quencher molecule. Insome embodiments the Quantitative PCR kits can also include componentssuitable for reverse-transcribing RNA including enzymes (e.g., reversetranscriptases such as AMV, MMLV and the like) and primers for reversetranscription along with deoxynucleotides and buffers needed for thereverse transcription reaction. Each component of the quantitative PCRkit is generally in its own suitable container. Thus, these kitsgenerally include distinct containers suitable for each individualreagent, enzyme, primer and probe. Further, the quantitative PCR kitscan include instructions for performing the assay and methods forinterpreting and analyzing the data resulting from the performance ofthe assay. In a specific embodiment, the kits contain instructions forpredicting whether a hematological cancer in a patient is clinicallysensitive to a compound.

For antibody based kits, the kit can include, for example: (1) a firstantibody which binds to a polypeptide or protein of interest; and,optionally, (2) a second, different antibody which binds to either thepolypeptide or protein, or the first antibody and is conjugated to adetectable label (e.g., a fluorescent label, radioactive isotope orenzyme). The first antibody can be attached to a solid support. In aspecific embodiment, the polypeptide or protein of interest is abiomarker provided herein. The antibody-based kits can also includebeads for conducting an immunoprecipitation. Each component of theantibody-based kits is generally in its own suitable container. Thus,these kits generally include distinct containers suitable for eachantibody. Further, the antibody-based kits can include instructions forperforming the assay and methods for interpreting and analyzing the dataresulting from the performance of the assay. In a specific embodiment,the kits contain instructions for predicting whether a hematologicalcancer in a patient is clinically sensitive to an FTI.

In some embodiments a kit provided herein includes an FTI providedherein, or a pharmaceutically composition having an FTI. Kits canfurther include additional active agents, including but not limited tothose disclosed herein, such as a DNA-hypomethylating agent, atherapeutic antibody that specifically binds to a cancer antigen, ahematopoietic growth factor, a cytokine, an anti-cancer agent, anantibiotic, a cox-2 inhibitor, an immunomodulatory agent, ananti-thymocyte globulin, an immunosuppressive agent, or acorticosteroid.

Kits provided herein can further include devices that are used toadminister the FTI or other active ingredients. Examples of such devicesinclude, but are not limited to, syringes, drip bags, patches, andinhalers.

Kits can further include cells or blood for transplantation as well aspharmaceutically acceptable vehicles that can be used to administer oneor more active ingredients. For example, if an active ingredient isprovided in a solid form that must be reconstituted for parenteraladministration, the kit can comprise a sealed container of a suitablevehicle in which the active ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

In certain embodiments of the methods and kits provided herein, solidphase supports are used for purifying proteins, labeling samples orcarrying out the solid phase assays. Examples of solid phases suitablefor carrying out the methods disclosed herein include beads, particles,colloids, single surfaces, tubes, multiwell plates, microtiter plates,slides, membranes, gels and electrodes. When the solid phase is aparticulate material (e.g., beads), it is, in one embodiment,distributed in the wells of multi-well plates to allow for parallelprocessing of the solid phase supports.

The kit of this disclosure can include an ancillary reagent. In someembodiments, the ancillary reagent can be a secondary antibody, adetection reagent, a detection buffer, an immobilization buffer, adilution buffer, a washing buffer, or any combination thereof.

Secondary antibodies can be monoclonal or polyclonal antibodies.Secondary antibodies can be derived from any mammalian organism,including bovine, mice, rats, hamsters, goats, camels, chicken, rabbit,and others. Secondary antibodies can include, for example, an anti-humanIgA antibody, an anti-human IgD antibody, an anti-human IgE antibody, ananti-human IgG antibody, or an anti-human IgM antibody. Secondaryantibodies can be conjugated to enzymes (e.g., horseradish peroxidase(HRP), alkaline phosphatase (AP), luciferase, and the like) or dyes(e.g., colorimetric dyes, fluorescent dyes, fluorescence resonanceenergy transfer (FRET)-dyes, time-resolved (TR)-FRET dyes, and thelike). In some embodiments, the secondary antibody is a polyclonalrabbit-anti-human IgG antibody, which is HRP-conjugated.

Any detection reagent known in the art can be included in a kit of thisdisclosure. In some embodiments, the detection reagent is a colorimetricdetection reagent, a fluorescent detection reagent, or achemiluminescent detection reagent. In some embodiments, thecolorimetric detection reagent includes PNPP (p-nitrophenyl phosphate),ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) or OPD(o-phenylenediamine). In some embodiments, the fluorescent detectionreagent includes QuantaBlu™ or QuantaRed™ (Thermo Scientific, Waltham,Mass.). In some embodiments, the luminescent detection reagent includesluminol or luciferin. In some embodiments, the detection reagentincludes a trigger (e.g., H2O2) and a tracer (e.g.,isoluminol-conjugate).

Any detection buffer known in the art can be included in a kit of thisdisclosure. In some embodiments the detection buffer is acitrate-phosphate buffer (e.g., about pH 4.2).

Any stop solution known in the art can be included in a kit of thisdisclosure. The stop solutions of this disclosure terminate or delay thefurther development of the detection reagent and corresponding assaysignals. Stop solutions can include, for example, low-pH buffers (e.g.,glycine-buffer, pH 2.0), chaotrophic agents (e.g., guanidinium chloride,sodium-dodecylsulfate (SDS)) or reducing agents (e.g., dithiothreitol,mecaptoethanol), or the like.

In some embodiments, the ancillary reagent is an immobilization reagent,which can be any immobilization reagent known in the art, includingcovalent and non-covalent immobilization reagents. Covalentimmobilization reagents can include any chemical or biological reagentthat can be used to covalently immobilize a peptide or a nucleic acid ona surface. Covalent immobilization reagents can include, for example, acarboxyl-to-amine reactive group (e.g., carbodiimides such as EDC orDCC), an amine reactive group (e.g., N-hydroxysuccinimide (NHS) esters,imidoesters), a sulfhydryl-reactive crosslinker (e.g., maleimides,haloacetyls, pyridyl disulfides), a carbonyl-reactive crosslinker groups(e.g., hydrazides, alkoxyamines), a photoreactive crosslinker (e.g.,aryl azides, dizirines), or a chemoselective ligation group (e.g., aStaudinger reaction pair). Non-covalent immobilization reagents includeany chemical or biological reagent that can be used to immobilize apeptide or a nucleic acid non-covalently on a surface, such as affinitytags (e.g., biotin) or capture ragents (e.g., streptavidin or anti-tagantibodies, such as anti-His6 (“His6” disclosed as SEQ ID NO: 50) oranti-Myc antibodies).

The kits of this disclosure can include combinations of immobilizationreagents. Such combinations include, for example, EDC and NHS, which canbe used, for example, to immobilize a protein of this disclosure on asurface, such as a carboxylated dextrane matrix (e.g., on a BIAcore™ CM5chip or a dextrane-based bead). Combinations of immobilization reagentscan be stored as premixed reagent combinations or with one or moreimmobilization reagents of the combination being stored separately fromother immobilization reagents.

A large selection of washing buffers are known in the art, such astris(hydroxymethyl)aminomethane (Tris)-based buffers (e.g.,Tris-buffered saline, TBS) or phosphate buffers (e.g.,phosphate-buffered saline, PBS). Washing buffers can include detergents,such as ionic or non-ionic detergents. In some embodiments, the washingbuffer is a PBS buffer (e.g., about pH 7.4) including Tween®20 (e.g.,about 0.05% Tween®20).

Any dilution buffer known in the art can be included in a kit of thisdisclosure. Dilution buffers can include a carrier protein (e.g., bovineserum albumin, BSA) and a detergent (e.g., Tween®20). In someembodiments, the dilution buffer is PBS (e.g., about pH 7.4) includingBSA (e.g., about 1% BSA) and Tween®20 (e.g., about 0.05% Tween®20).

In some embodiments, the kit of this disclosure includes a cleaningreagent for an automated assay system. An automated assay system caninclude systems by any manufacturer. In some embodiments, the automatedassay systems include, for example, the BIO-FLASH™, the BEST 2000™, theDS2™, the ELx50 WASHER, the ELx800 WASHER, and the ELx800 READER. Acleaning reagent can include any cleaning reagent known in the art.

It is noted that any combination of the above-listed embodiments, forexample, with respect to one or more reagents, such as, withoutlimitation, nucleic acid primers, solid support and the like, are alsocontemplated in relation to any of the various methods and/or kitsprovided herein.

4. WILD TYPE K-RAS AND N-RAS AS BIOMARKERS FOR FTI TREATMENT

Provided herein are methods of selection of cancer patients fortreatment with an FTI are based, in part, on the discovery that themutation status in Ras is associated with clinical benefits of FTI, andcan be used to predict the responsiveness of a cancer patient to an FTItreatment. Accordingly, provided herein are methods for predictingresponsiveness of a cancer patient to an FTI treatment, methods forcancer patient population selection for an FTI treatment, and methodsfor treating cancer in a subject with a therapeutically effective amountof an FTI, based on the mutation status of Ras in a sample from thepatient.

4.1. RAS MUTATION STATUS

In some embodiments, provided herein is a method of treating a cancer ina subject based on the mutation status of K-Ras, N-Ras, or both. Themethod provided herein includes (a) determining the presence or absenceof a Ras mutation in a sample from the subject, wherein the Ras mutationincludes a K-Ras mutation or a N-Ras mutation, and subsequently (b)administering a therapeutically effective amount of an FTI to saidsubject if said sample is determined to lack the K-Ras mutation or theN-Ras mutation.

In some embodiments, the method provided herein includes (a) determiningthe presence or absence of a K-Ras mutation in a sample from thesubject, and subsequently (b) administering a therapeutically effectiveamount of an FTI to said subject if said sample is determined to lackthe K-Ras mutation. In some embodiments, the sample is determined tohave wild type K-Ras.

In some embodiments, the method provided herein includes (a) determiningthe presence or absence of a N-Ras mutation in a sample from thesubject, and subsequently (b) administering a therapeutically effectiveamount of an FTI to said subject if said sample is determined to lackthe N-Ras mutation. In some embodiments, the sample is determined tohave wild type N-Ras.

In some embodiments, the K-Ras mutation is K_(A)-Ras mutation. In someembodiments, the K-Ras mutation is K_(B)-Ras mutation. In someembodiments, the K-Ras mutation is a combination of K_(A)-Ras mutationand a K_(B)-Ras mutation. The K-Ras mutation can include a mutation at acodon selected from the group consisting of G12, G13, and Q61 ofK_(A)-Ras, K_(B)-Ras, or both. In some embodiments, the K_(A)-Rasmutation can include a mutation selected from the group consisting ofthe amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R,G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R andA146V. In some embodiments, the K_(B)-Ras mutation can include amutation selected from the group consisting of the amino acidsubstitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C,G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the Ras mutation is an N-Ras mutation. In someembodiments, the N-Ras mutation can include at least one mutation at acodon selected from the group consisting of G12, G13, G15, G60 and Q61.In some embodiments, the N-Ras mutation can include at least onemutation at a codon selected from the group consisting of G12, G13, andQ61. In some embodiments, the N-Ras mutation can include at least onemutation selected from the group consisting of the amino acidsubstitutions of G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R,G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the sample is determined to not have amino acidsubstitution at G12, G13, and Q61 of K-Ras, and also not have amino acidsubstitution at G12, G13, and Q61 of N-Ras. In some embodiments, thesample is determined to not have any K-Ras mutation or any N-Rasmutation. In some embodiments, the sample is determined to have wildtype K-Ras and wild type N-Ras.

In some embodiments, the method provided herein further includesdetermining the presence or absence of an H-Ras mutation in a samplefrom the subject, and administering a therapeutically effective amountof an FTI to said subject if said sample is determined to have an H-Rasmutation.

In some embodiments, the H-Ras mutation is a mutation at a codonselected from the group consisting of G12, G13, and Q61. In someembodiments, the H-Ras mutation can be a mutation selected from thegroup consisting of the amino acid substitutions of G12R, G12V, G13C,G13R, Q61L and Q61R.

In some embodiments, provided herein is a method of treating a cancer ina subject based on the mutation status of K-Ras and N-Ras, whichincludes (a) determining the presence or absence of a K-Ras mutation anda N-Ras mutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample does not have any K-Ras mutation or any N-Rasmutation. In some embodiment, the method includes administering atherapeutically effective amount of an FTI to the subject if the samplehas wild type K-Ras and wild type N-Ras. In some embodiment, the methodfurther includes determining the mutation status of H-Ras, andsubsequently administering a therapeutically effective amount of an FTIto the subject if the sample of the subject does not have any K-Rasmutation or any N-Ras mutation, but has a H-Ras mutation.

Provided herein are methods for predicting responsiveness of a cancerpatient to an FTI treatment, methods for cancer patient populationselection for an FTI treatment, and methods for treating cancer in asubject with a therapeutically effective amount of an FTI, based on themutation status of Ras in a sample from the patient. In someembodiments, the method includes determining the presence or absence ofa Ras mutation in a sample from the subject prior to beginningtreatment. Tumors or cancers that do not have K-Ras mutation or N-Rasmutation indicate that the patients will likely be responsive to the FTItreatment. In some embodiments, patients are selected for FTI treatmentbased on the lack of K-Ras mutation or N-Ras mutation. In someembodiments, patients are selected for FTI treatment based on the lackof K-Ras mutation and N-Ras mutation. In some embodiments, patients arefurther selected based on the presence of H-Ras mutation. The mutationstatus of Ras can be detected at the nucleic acid or protein level. Insome embodiments, the Ras mutation status is determined by analyzingnucleic acids obtained from the sample. In some embodiments, the Rasmutation status is determined by analyzing protein obtained from thesample.

Techniques can be used in methods provided herein include in situhybridization (Stoler, Clin. Lab. Med. 12:215-36 (1990), usingradioisotope or fluorophore-labeled probes; polymerase chain reaction(PCR); quantitative Southern blotting, dot blotting and other techniquesfor quantitating individual genes. In some embodiments, probes orprimers selected for gene amplification evaluation are highly specificto avoid detecting closely related homologous genes. Alternatively,antibodies can be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn can be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

In some embodiments, the Ras mutation status is determined by analyzingnucleic acids obtained from the sample. The nucleic acids may be mRNA orgenomic DNA molecules from the test subject. Methods for determining Rasmutation status by analyzing nucleic acids are well known in the art. Insome embodiments, the methods include sequencing, Polymerase ChainReaction (PCR), DNA microarray, Mass Spectrometry (MS), SingleNucleotide Polymorphism (SNP) assay, denaturing high-performance liquidchromatography (DHPLC), or Restriction Fragment Length Polymorphism(RFLP) assay. In some embodiments, the Ras mutation status is determinedusing standard sequencing methods, including, for example, Sangersequencing, next generation sequencing (NGS). In some embodiments, theRas mutation status is determined using MS.

In some embodiments, the method includes determining the presence orabsence of a Ras mutation by amplifying Ras nucleic acid from a sampleby PCR. For example, PCR technology and primer pairs that can be usedare known to the person skilled in the art. (e.g., Chang et al.,Clinical Biochemistry, 43 (2010), 296-301; WO2015144184). For example, amultiplex PCR can be used to amplify codons 12 and 13 of exon 2 andcodon 61 of exon 3 of N-, H-, or K-Ras genes with two pairs of universalprimers for exons 2 and 3. For example, the following primers can beused:

SEQ ID NO Exon Primer Sequence 21 25′-CYKRBKDRMRATGACKGARTAYAARCTKGTGGT-3′ 22 25′-ACCTCTATDGTKGGRTCRTATTC-3′ 23 3 5′-CAGGATTCYTACMGRAARCARGT-3′ 24 45′-TTKATGGCAAAYACACAVAGRAAGC-3′

As used herein, the letters are used according to the IUPAC notation,e.g. “Y” denotes pyrimidine, “K” denotes keto, e.g. G or C, “R” denotespurine, “B” C, G, or T, “D” denotes A, G, or T, “M” denotes A, C, “V”denotes A, C, or G.

Following multiplex PCR amplification, the products can be purified toremove the primers and unincorporated deoxynucleotide triphosphatesusing PCR-M™ Clean Up System (Viogenebiotek Co., Sunnyvale, Calif.,USA). Purified DNA can then be semiquantified on a 1% agarose gel in0.5×TBE and visualized by staining with ethidium bromide. The productscan then be subjected to primer extension analysis using primers asdisclosed in Chang et al., Clinical Biochemistry 43 (2010), 296-301,e.g., such as the following:

SEQ ID NO RAS Primer Sequence 25 K 5′-AACTTGTGGTAGTTGGAGCT 26 K5′-ACTGAATATAAACTTGTGGTAGTTGGAGCTG 27 K 5′-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGT 28 K 5′-GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG 29 K 5′-GCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGT 30 K 5′-GGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTC 31 K 5′-T₄₅ATTCTCGACACAGCAGGTCA 32 N5′-AACTGGTGGTGGTTGGAGCA-3′ 33 N 5′-T₇AACTGGTGGTGGTTGGAGCAG-3′ 34 N5′-T₁₄CAGTGCGCTTTTCCCAACAC-3′ 35 N 5′-T₂₂GTGGTGGTTGGAGCAGGTG-3′ 36 N5′-T₂₉CTCATGGCACTGTACTCTTCTT-3′ 37 N 5′-T₃₆CTCATGGCACTGTACTCTTCT-3′ 38 N5′-T₄₃CTCTCATGGCACTGTACTCTTC-3′ 39 H 5′-AGCTGGTGGTGGTGGGCGCC-3′ 40 H5′-T₇AGCTGGTGGTGGTGGGCGCCG-3′ 41 H 5′-T₁₄TGGTGGTGGTGGGCGCCGGC-3′ 42 H5′-T₂₂GTGGTGGTGGGCGCCGGCG-3′ 43 H 5′-T₂₉ACATCCTGGATACCGCCGGC-3′ 44 H5′-T₃₆ACATCCTGGATACCGCCGGCC-3′ 45 H 5′-T₄₃CGCATGGCGCTGTACTCCTC-3′

Various concentrations of probe for either codon 12, 13, or 61 can beemployed (e.g. 0.03-0.6 μM) in the reactions containing 1.5 μl ofpurified PCR products, as well as 4 μI of ABI PRISM SNaPshot MultiplexKit (Applied Biosystems, Foster City, Calif.) containing AmpliTaq® DNApolymerase and fluorescently labeled dideoxynucleotide triphosphates(ddNTPs) (RGG-labeled dideoxyadenosine triphosphate, TAMRA-labeleddideoxycytidine triphosphate, ROX-labeled dideoxythymidine triphosphate,and R1 10-labeled dideoxyguanosine triphosphate). Each 10-μI mixture canthen be subjected to 25 single-base extension cycles consisting of adenaturing step at 96° C. for 10 s and primer annealing and extension at55° C. for 35 s. After cycle extension, unincorporated fluorescentddNTPs can then be incubated with 1 μI of shrimp alkaline phosphatase(United States Biochemical Co., Cleveland, USA) at 37° C. for 1 h,followed by enzyme deactivation at 75° C. for 15 min. The primerextension reaction products can then be resolved by automated capillaryelectrophoresis on a capillary electrophoresis platform, e.g. 14 μl ofHi-Di™ Formamide (Applied Biosystems) and 0.28 μl of GeneScan™-120LIZ®Size Standard (Applied Biosystems) were added to 6 μI of primerextension products. All samples may then e.g. be analyzed on an ABIPrism 310 DNA Genetic Analyzer (Applied Biosystems) according tomanufacturer's instructions using GeneScan™ 3.1 (Applied Biosystems).

Provided herein are methods of selecting a cancer patient who is likelyto benefit from an FTI treatment, include determining the presence orabsence of a Ras mutation by amplifying Ras nucleic acid from thepatient's tumor sample and sequencing the amplified nucleic acid.Accordingly, Ras nucleic acid can be amplified using primers asdisclosed above and sequenced. For example, K-Ras, N-Ras and H-Rasnucleic acid can be amplified by PCR as disclosed above and subsequentlysubcloned using e.g. the TOPO TA Cloning Kit for sequencing(Invitrogen).

In the above inventive method, RAS nucleic acid can be obtained from thepatient's tumor sample by any method known to the person skilled in theart. For example, any commercial kit may be used to isolate the genomicDNA, or mRNA from a tumor sample, such as e.g. the Qlamp DNA mini kit,or RNeasy mini kit (Qiagen, Hilden, Germany). For example, if mRNA wasisolated from the patient's tumor sample, cDNA synthesis can be carriedout prior to the methods as disclosed herein, according to any knowntechnology in the art.

For example, the nucleic acid to be isolated from a tumor can forexample be one of genomic DNA, total RNA, mRNA or poly(A)+ mRNA. Forexample, if mRNA has been isolated from the the patient's tumor sample,the mRNA (total mRNA or poly(A)+ mRNA) may be used for cDNA synthesisaccording to well established technologies in prior art, such as thoseprovided in commercial cDNA synthesis kits, e.g. Superscript® III FirstStrand Synthesis Kit. The cDNA can then be further amplified by means ofe.g. PCR and subsequently subjected to sequencing by e.g. Sangersequencing or pyro-sequencing to determine the nucleotide sequence ofe.g. codons 12 and 13 of the RAS gene, e.g. H-RAS, N-RAS or KRAS.Alternatively, the PCR product can e.g. also be subcloned into a TA TOPOcloning vector for sequencing. Other technologies than sequencing todetermine the absence or presence of Ras mutations can be used in themethods provided herein such as e.g. Single Nucleotide Primer Extension(SNPE) (PLoS One. 2013 Aug. 21; 8(8):e72239); DNA microarray, MassSpectrometry (MS) (e.g. matrix-assisted laser desorption/ionizationtime-of-flight (MALDI-TOF) mass spectrometry), Single NucleotidePolymorphism (SNP), denaturing high-performance liquid chromatography(DHPLC), or Restriction Fragment Length Polymorphism (RFLP) assay.

For example, Single Nucleotide Polymorphism (SNP) Assay can be used fordetermining the Ras mutation status in a sample. The SNP assay can beperformed on the HT7900 from Applied Biosystems, following the allelicdiscrimination assay protocol provided by the manufacturer. Ras mutationstatus can also be determined by DHPLC or RFLP, or any other methodsknown in the art. Bowen et al., Blood, 106:2113-2119 (2005); Bowen etal., Blood, 101:2770-2774 (2003); Nishikawa et al., Clin Chim Acta.,318:107-112 (2002); Lin S Y et al., Am J Clin Pathol. 100:686-689(1993); O'Leary J J et al., J Clin Pathol. 51:576-582 (1998).

In some embodiments, the Ras mutation status is determined by analyzingprotein obtained from the sample. The mutated Ras protein can bedetected by a variety of immunohistochemistry (IHC) approaches or otherimmunoassay methods known in the art. IHC staining of tissue sectionshas been shown to be a reliable method of assessing or detectingpresence of proteins in a sample. Immunohistochemistry techniquesutilize an antibody to probe and visualize cellular antigens in situ,generally by chromogenic or fluorescent methods. Thus, antibodies orantisera, preferably polyclonal antisera, and most preferably monoclonalantibodies that specifically target mutant K-Ras or N-Ras can be used todetect expression. As discussed in greater detail below, the antibodiescan be detected by direct labeling of the antibodies themselves, forexample, with radioactive labels, fluorescent labels, hapten labels suchas, biotin, or an enzyme such as horse radish peroxidase or alkalinephosphatase. Alternatively, unlabeled primary antibody is used inconjunction with a labeled secondary antibody, comprising antisera,polyclonal antisera or a monoclonal antibody specific for the primaryantibody. Immunohistochemistry protocols and kits are well known in theart and are commercially available. Automated systems for slidepreparation and IHC processing are available commercially. The Ventana®BenchMark XT system is an example of such an automated system.

Standard immunological and immunoassay procedures can be found in Basicand Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover,the immunoassays can be performed in any of several configurations,which are reviewed extensively in Enzyme Immunoassay (Maggio, ed.,1980); and Harlow & Lane, supra. For a review of the generalimmunoassays, see also Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Ten, eds., 7th ed. 1991).

Assays to detect K-Ras mutations or N-Ras mutations includenoncompetitive assays, e.g., sandwich assays, and competitive assays.Typically, an assay such as an ELISA assay can be used. ELISA assays areknown in the art, e.g., for assaying a wide variety of tissues andsamples, including blood, plasma, serum or bone marrow.

A wide range of immunoassay techniques using such an assay format areavailable, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and4,018,653, which are hereby incorporated by reference in theirentireties. These include both single-site and two-site or “sandwich”assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. These assays also include direct binding ofa labeled antibody to a target mutant Ras protein. Sandwich assays arecommonly used assays. A number of variations of the sandwich assaytechnique exist. For example, in a typical forward assay, an unlabelledantibody is immobilized on a solid substrate, and the sample to betested brought into contact with the bound molecule. After a suitableperiod of incubation, for a period of time sufficient to allow formationof an antibody-antigen complex, a second antibody specific to theantigen, labeled with a reporter molecule capable of producing adetectable signal is then added and incubated, allowing time sufficientfor the formation of another complex of antibody-antigen-labeledantibody. Any unreacted material is washed away, and the presence of theantigen is determined by observation of a signal produced by thereporter molecule. The results may either be qualitative, by simpleobservation of the visible signal, or may be quantitated by comparingwith a control sample.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labeled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for themutant Ras protein is either covalently or passively bound to a solidsurface. The solid surface may be glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride, or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated for aperiod of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g., from room temperatureto 40° C. such as between 25° C. and 32° C. inclusive) to allow bindingof any subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the mutant Ras protein. Thesecond antibody is linked to a reporter molecule which is used toindicate the binding of the second antibody to the mutant Ras protein.

In some embodiments, flow cytometry (FACS) can be used to detect themutant K-Ras or N-Ras using antibodies specific target the mutant K-Rasor N-Ras. The flow cytometer detects and reports the intensity of thefluorichrome-tagged antibody, which indicates the presence of the mutantK-Ras or N-Ras. Non-fluorescent cytoplasmic proteins can also beobserved by staining permeablized cells. The stain can either be afluorescence compound able to bind to certain molecules, or afluorichrome-tagged antibody to bind the molecule of choice.

An alternative method involves immobilizing the target Ras protein inthe sample and then exposing the immobilized target to mutant specificantibody which may or may not be labeled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target can be detectable by direct labelingwith the antibody. Alternatively, a second labeled antibody, specific tothe first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by a labeled reportermolecule.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase, and alkaline phosphatase, and other arediscussed herein. The substrates to be used with the specific enzymesare generally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. Examples of suitableenzymes include alkaline phosphatase and peroxidase. It is also possibleto employ fluorogenic substrates, which yield a fluorescent productrather than the chromogenic substrates noted above. In all cases, theenzyme-labeled antibody is added to the first antibody-molecular markercomplex, allowed to bind, and then the excess reagent is washed away. Asolution containing the appropriate substrate is then added to thecomplex of antibody-antigen-antibody. The substrate will react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of mutantRas protein which was present in the sample. Alternately, fluorescentcompounds, such as fluorescein and rhodamine, can be chemically coupledto antibodies without altering their binding capacity. When activated byillumination with light of a particular wavelength, thefluorochrome-labeled antibody adsorbs the light energy, inducing a stateto excitability in the molecule, followed by emission of the light at acharacteristic color visually detectable with a light microscope. As inthe EIA, the fluorescent labeled antibody is allowed to bind to thefirst antibody-molecular marker complex. After washing off the unboundreagent, the remaining tertiary complex is then exposed to the light ofthe appropriate wavelength, the fluorescence observed indicates thepresence of the molecular marker of interest. Immunofluorescence and EIAtechniques are both very well established in the art and are discussedherein.

In some embodiments, the determination of the Ras mutation status isperformed as a companion diagnostic to the FTI treatment. The companiondiagnostic can be performed at the clinic site where the subject istreated. The companion diagnostic can also be performed at a siteseparate from the clinic site where the subject is treated.

As a person of ordinary skill in the art would understand, methodsprovided herein are for predicting responsiveness of a cancer patient toan FTI treatment, methods for cancer patient population selection for anFTI treatment, and methods for treating cancer in a subject with atherapeutically effective amount of an FTI, based on the mutation statusof Ras in a sample from the patient. Any methods described herein orotherwise known in the art for determining the mutation status of Rascan be applied in the methods.

4.2. SAMPLES

In some embodiments, methods provided herein include obtaining a samplefrom the subject. The sample used in the methods provided hereinincludes body fluids from a subject. Non-limiting examples of bodyfluids include blood (e.g., peripheral whole blood, peripheral blood),blood plasma, bone marrow, amniotic fluid, aqueous humor, bile, lymph,menses, serum, urine, cerebrospinal fluid surrounding the brain and thespinal cord, synovial fluid surrounding bone joints.

In one embodiment, the sample is a bone marrow sample. Procedures toobtain a bone marrow sample are well known in the art, including but notlimited to bone marrow biopsy and bone marrow aspiration. Bone marrowhas a fluid portion and a more solid portion. In bone marrow biopsy, asample of the solid portion is taken. In bone marrow aspiration, asample of the fluid portion is taken. Bone marrow biopsy and bone marrowaspiration can be done at the same time and referred to as a bone marrowexam.

In some embodiments, the sample is a blood sample. The blood sample canbe obtained using conventional techniques as described in, e.g. Innis etal, editors, PCR Protocols (Academic Press, 1990). White blood cells canbe separated from blood samples using convention techniques orcommercially available kits, e.g. RosetteSep kit (Stein CellTechnologies, Vancouver, Canada). Sub-populations of white blood cells,e.g. mononuclear cells, NK cells, B cells, T cells, monocytes,granulocytes or lymphocytes, can be further isolated using conventionaltechniques, e.g. magnetically activated cell sorting (MACS) (MiltenyiBiotec, Auburn, Calif.) or fluorescently activated cell sorting (FACS)(Becton Dickinson, San Jose, Calif.).

In one embodiment, the blood sample is from about 0.1 mL to about 10.0mL, from about 0.2 mL to about 7 mL, from about 0.3 mL to about 5 mL,from about 0.4 mL to about 3.5 mL, or from about 0.5 mL to about 3 mL.In another embodiment, the blood sample is about 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0,8.0, 9.0 or 10.0 mL.

In some embodiments, the sample used in the present methods includes abiopsy (e.g., a tumor biopsy). The biopsy can be from any organ ortissue, for example, skin, liver, lung, heart, colon, kidney, bonemarrow, teeth, lymph node, hair, spleen, brain, breast, or other organs.Any biopsy technique known by those skilled in the art can be used forisolating a sample from a subject, for instance, open biopsy, closebiopsy, core biopsy, incisional biopsy, excisional biopsy, or fineneedle aspiration biopsy.

In certain embodiments, the sample used in the methods provided hereinincludes a plurality of cells. Such cells can include any type of cells,e.g., stem cells, blood cells (e.g., PBMCs), lymphocytes, NK cells, Bcells, T cells, monocytes, granulocytes, immune cells, or tumor orcancer cells. Specific cell populations can be obtained using acombination of commercially available antibodies (e.g., Quest Diagnostic(San Juan Capistrano, Calif.); Dako (Denmark)).

In certain embodiments, the sample used in the methods provided hereinis from a diseased tissue, e.g., from an individual having cancer (e.g.,lymphoma, MDS, or leukemia). In certain embodiments. In someembodiments, the cells can be obtained from the tumor or cancer cells ora tumor tissue, such as a tumor biopsy or a tumor explants. In certainembodiments, the number of cells used in the methods provided herein canrange from a single cell to about 10⁹ cells. In some embodiments, thenumber of cells used in the methods provided herein is about 1×10⁴,5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, or 5×10⁸.

The number and type of cells collected from a subject can be monitored,for example, by measuring changes in morphology and cell surface markersusing standard cell detection techniques such as flow cytometry, cellsorting, immunocytochemistry (e.g., staining with tissue specific orcell-marker specific antibodies) fluorescence activated cell sorting(FACS), magnetic activated cell sorting (MACS), by examination of themorphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. Fluorescence activated cell sorting (FACS) is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In certain embodiments, subsets of cells are used in the methodsprovided herein. Methods to sort and isolate specific populations ofcells are well-known in the art and can be based on cell size,morphology, or intracellular or extracellular markers. Such methodsinclude, but are not limited to, flow cytometry, flow sorting, FACS,bead based separation such as magnetic cell sorting, size-basedseparation (e.g., a sieve, an array of obstacles, or a filter), sortingin a microfluidics device, antibody-based separation, sedimentation,affinity adsorption, affinity extraction, density gradientcentrifugation, laser capture microdissection, etc.

The sample can be a whole blood sample, a bone marrow sample, apartially purified blood sample, or PBMC. The sample can be a tissuebiopsy or a tumor biopsy. In some embodiments, the sample is a bonemarrow sample from a cancer patient. In some embodiments, the sample isPBMCs from a cancer patient.

4.3 CANCERS

Provided herein are methods to treat a cancer in a subject with an FTI,and methods for selecting cancer patients for an FTI treatment based onthe lack of K-Ras mutation and N-Ras mutation. The cancer can be ahematopoietic cancer or a solid tumor. Provided herein are also methodsto treat a premalignant condition in a subject with an FTI, and methodsfor selecting patients with a premalignant condition for an FTItreatment based on the lack of K-Ras mutation and N-Ras mutation.

In some embodiments, provided herein are methods to treat a solid tumorwith an FTI based on the lack of K-Ras mutation and N-Ras mutation.Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). The solid tumorto be treated with the methods of the invention can be sarcomas andcarcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, pheochromocytomas sebaceous gland carcinoma, papillarycarcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma(such as brainstem glioma and mixed gliomas), glioblastoma (also knownas glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,meduloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases). In someembodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods to treat a solid tumorwith an FTI based on the lack of K-Ras mutation and N-Ras mutation,wherein the solid tumor is malignant melanoma, adrenal carcinoma, breastcarcinoma, renal cell cancer, carcinoma of the pancreas, non-small-celllung carcinoma (NSCLC) or carcinoma of unknown primary. In someembodiments, the FTI is tipifarnib. Drugs commonly administered topatients with various types or stages of solid tumors include, but arenot limited to, celebrex, etoposide, cyclophosphamide, docetaxel,apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In some embodiments, the solid tumor to be treated by methods providedherein can be thyroid cancer, head and neck cancers, urothelial cancers,salivary cancers, cancers of the upper digestive tract, bladder cancer,breast cancer, ovarian cancer, brain cancer, gastric cancer, prostatecancer, lung cancer, colon cancer, skin cancer, liver cancer, andpancreatic cancer. In some embodiments, the bladder cancer to be treatedby methods provided herein can be transitional cell carcinoma. In someembodiments, the FTI is tipifarnib.

In some embodiments, the solid tumor to be treated by methods providedherein can be selected from the groups consisting of carcinoma,melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the premalignant conditions to be treated bymethods provided herein can be actinic cheilitis, Barrett's esophagus,atrophic gastritis, ductal carcinoma in situ, Dyskeratosis congenita,Sideropenic dysphagia, Lichen planus, Oral submucous fibrosis, Solarelastosis, cervical dysplasia, polyps, leukoplakia, erythroplakia,squamous intraepithelial lesion, a pre-malignant disorder, or apre-malignant immunoproliferative disorder.

In some embodiments, provided herein are methods to treat ahematopoietic cancer in a subject with an FTI or selecting cancerpatients for an FTI treatment based on the lack of K-Ras mutation andN-Ras mutation. Hematologic cancers are cancers of the blood or bonemarrow. Examples of hematological (or hematogenous) cancers includemyeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS),leukemia, and lymphoma. In some embodiments, the cancer is acute myeloidleukemia (AML), natural killer cell lymphoma (NK lymphoma), naturalkiller cell leukemia (NK leukemia), cutaneous T-Cell lymphoma (CTCL),juvenile myelomonocytic leukemia (JMML), peripheral T-cell lymphoma(PTCL), chronic myeloid leukemia (CML) or chronic myelomonocyticleukemia (CMML). In some embodiments, the cancer is CMML. In someembodiments, the cancer is JMML.

In some embodiments, provided herein are methods to treat CMML in asubject with an FTI or selecting CMML patients for an FTI treatmentbased on the lack of K-Ras mutation and N-Ras mutation. CMML isclassified as a myelodysplastic/myeloproliferative neoplasm by the 2008World Health Organization classification of hematopoietic tumors. TheCMML can be myelodysplastic CMML or myeloproliferative CMML. CMMLpatients have a high number of monocytes in their blood (at least 1,000per mm³). Two classes-myelodysplastic and myeloproliferative-have beendistinguished upon the level of the white blood cell count (threshold 13G/L). Often, the monocyte count is much higher, causing their totalwhite blood cell count to become very high as well. Usually there areabnormal cells in the bone marrow, but the amount of blasts is below20%. About 15% to 30% of CMML patients go on to develop acute myeloidleukemia. The diagnosis of CMML rests on a combination of morphologic,histopathologic and chromosomal abnormalities in the bone marrow. TheMayo prognostic model classified CMML patients into three risk groupsbased on: increased absolute monocyte count, presence of circulatingblasts, hemoglobin <10 gm/dL and platelets <100×10⁹/L. The mediansurvival was 32 months, 18.5 months and 10 months in the low,intermediate, and high-risk groups, respectively. The Groupe Francophonedes (GFM) score segregated CMML patients into three risk groups basedon: age >65 years, WBC >15×10⁹/L, anemia, platelets <100×10⁹/L, andASXL1 mutation status. After a median follow-up of 2.5 years, survivalranged from not reached in the low-risk group to 14.4 months in thehigh-risk group.

In some embodiments, provided herein are methods of treating CMML in asubject by determining the presence or absence of a K-Ras mutation and aN-Ras mutation in a sample from the subject, and subsequentlyadministering a therapeutically effective amount of the FTI to thesubject if the sample is determined to lack a K-Ras mutation and to lackN-Ras mutation. In some embodiments, the FTI is tipifarnib. In someembodiments, the sample is determined to have wild type K-Ras and wildtype N-Ras.

In some embodiments, provided herein are methods of treating CMML in asubject by determining the presence or absence of a K-Ras mutation in asample from the subject, and subsequently administering atherapeutically effective amount of the FTI to the subject if the sampleis determined to lack the K-Ras mutation. In some embodiments, the FTIis tipifarnib. In some embodiments, the sample is determined to havewild type K-Ras.

In some embodiments, provided herein are methods of treating CMML in asubject by determining the presence or absence of a N-Ras mutation in asample from the subject, and subsequently administering atherapeutically effective amount of the FTI to the subject if the sampleis determined to lack the N-Ras mutation. In some embodiments, the FTIis tipifarnib. In some embodiments, the sample is determined to havewild type N-Ras.

In some embodiments, provided herein are methods of treating CMML in asubject by determining the presence or absence of a K-Ras mutation and aN-Ras mutation in a sample from the subject, and subsequentlyadministering tipifarnib to the subject if the sample is determined tohave wild type K-Ras and wild type N-Ras.

In some embodiments, provided herein are methods to treat MDS in asubject with an FTI or selecting MDS patients for an FTI treatment. MDSrefers to a diverse group of hematopoietic stem cell disorders. MDS canbe characterized by a cellular marrow with impaired morphology andmaturation (dysmyelopoiesis), ineffective blood cell production, orhematopoiesis, leading to low blood cell counts, or cytopenias(including anemia, leukopenia, and thrombocytopenia), and high risk ofprogression to acute myeloid leukemia, resulting from ineffective bloodcell production. See The Merck Manual 953 (17th ed. 1999) and List etal., 1990, J Clin. Oncol. 8:1424.

MDS can be divided into a number of subtypes depending on at least 1)whether increased numbers of blast cells are present in bone marrow orblood, and what percentage of the marrow or blood is made up of theseblasts; 2) whether the marrow shows abnormal growth (dysplasia) in onlyone type of blood cell (unilineage dysplasia) or in more than one typeof blood cell (multilineage dysplasia); and 3) whether there arechromosome abnormalities in marrow cells and, if so, which type or typesof abnormalities. MDS can also categorized based on the surface markersof the cancer cells. According to the World Health Organization, MDSsubtypes include refractory cytopenia with unilineage dysplasia (RCUD),also known as refractory anemia, refractory neutropenia, or refractorythrombocytopenia; refractory anemia with ring sideroblasts (RARS);refractory cytopenia with multilineage dysplasia (RCMD), which includesRCMD-RS if multilineage dysplasia and ring sideroblasts both arepresent; refractory anemia with excess blasts-1 (RAEB-1) and refractoryanemia with excess blasts-2 (RAEB-2) (These subtypes mean that thepatients have at least 5 percent (RAEB-1) or at least 10 percent(RAEB-2) but less than 20 percent blasts in their marrow); MDSassociated with isolated abnormality of chromosome 5 [del(5q)]; andunclassifiable MDS (MDS-U).

As a group of hematopoietic stem cell malignancies with significantmorbidity and mortality, MDS is a highly heterogeneous disease, and theseverity of symptoms and disease progression can vary widely amongpatients. The current standard clinical tool to evaluate riskstratification and treatment options is the revised InternationalPrognostic Scoring System, or IPSS-R. The IPSS-R differentiates patientsinto five risk groups (Very Low, Low, Intermediate, High, Very High)based on evaluation of cytogenetics, percentage of blasts(undifferentiated blood cells) in the bone marrow, hemoglobin levels,and platelet and neutrophil counts. The WHO also suggested stratifyingMDS patients by a del (5q) abnormality.

According to the ACS, the annual incidence of MDS is approximately13,000 patients in the United States, the majority of which are 60 yearsof age or older. The estimated prevalence is over 60,000 patients in theUnited States. Approximately 75% of patients fall into the IPSS-R riskcategories of Very Low, Low, and Intermediate, or collectively known aslower risk MDS.

The initial hematopoietic stem cell injury can be from causes such as,but not limited to, cytotoxic chemotherapy, radiation, virus, chemicalexposure, and genetic predisposition. A clonal mutation predominatesover bone marrow, suppressing healthy stem cells. In the early stages ofMDS, the main cause of cytopenias is increased programmed cell death(apoptosis). As the disease progresses and converts into leukemia, genemutation rarely occurs and a proliferation of leukemic cells overwhelmsthe healthy marrow. The disease course differs, with some cases behavingas an indolent disease and others behaving aggressively with a veryshort clinical course that converts into an acute form of leukemia.

An international group of hematologists, the French-American-British(FAB) Cooperative Group, classified MDS disorders into five subgroups,differentiating them from AML. The Merck Manual 954 (17^(th) ed. 1999);Bennett J. M., et al., Ann. Intern. Med. 1985 October, 103(4): 620-5;and Besa E. C., Med. Clin. North Am. 1992 May, 76(3): 599-617. Anunderlying trilineage dysplastic change in the bone marrow cells of thepatients is found in all subtypes.

There are two subgroups of refractory anemia characterized by fivepercent or less myeloblasts in bone marrow: (1) refractory anemia (RA)and; (2) RA with ringed sideroblasts (RARS), defined morphologically ashaving 15% erythroid cells with abnormal ringed sideroblasts, reflectingan abnormal iron accumulation in the mitochondria. Both have a prolongedclinical course and low incidence of progression to acute leukemia. BesaE. C., Med. Clin. North Am. 1992 May, 76(3): 599-617.

There are two subgroups of refractory anemias with greater than fivepercent mycloblasts: (1) RA with excess blasts (RAEB), defined as 6-20%myeloblasts, and (2) RAEB in transformation (RAEB-T), with 21-30%myeloblasts. The higher the percentage of myeloblasts, the shorter theclinical course and the closer the disease is to acute myelogenousleukemia. Patient transition from early to more advanced stagesindicates that these subtypes are merely stages of disease rather thandistinct entities. Elderly patients with MDS with trilineage dysplasiaand greater than 30% myeloblasts who progress to acute leukemia areoften considered to have a poor prognosis because their response rate tochemotherapy is lower than de novo acute myeloid leukemia patients. Thefifth type of MDS, the most difficult to classify, is CMML. This subtypecan have any percentage of myeloblasts but presents with a monocytosisof 1000/dL or more. It may be associated with splenomegaly. This subtypeoverlaps with a myeloproliferative disorder and may have an intermediateclinical course. It is differentiated from the classic CML that ischaracterized by a negative Ph chromosome.

MDS is primarily a disease of elderly people, with the median onset inthe seventh decade of life. The median age of these patients is 65years, with ages ranging from the early third decade of life to as oldas 80 years or older. The syndrome may occur in any age group, includingthe pediatric population. Patients who survive malignancy treatment withalkylating agents, with or without radiotherapy, have a high incidenceof developing MDS or secondary acute leukemia. About 60-70% of patientsdo not have an obvious exposure or cause for MDS, and are classified asprimary MDS patients.

In some embodiments, provided herein are methods to treat MPN in asubject with an FTI or selecting MPN patients for an FTI treatment. MPNis a group of diseases that affect blood-cell formation. In all forms ofMPN, stem cells in the bone marrow develop genetic defects (calledacquired defects) that cause them to grow and survive abnormally. Thisresults in unusually high numbers of blood cells in the bone marrow(hypercellular marrow) and in the bloodstream. Sometimes in MPN, theabnormal stem cells cause scarring in the marrow, called myelofibrosis.Myelofibrosis can lead to low levels of blood cells, especially lowlevels of red blood cells (anemia). In MPN, the abnormal stem cells canalso grow in the spleen, causing the spleen to enlarge (splenomegaly),and in other sites outside the marrow, causing enlargement of otherorgans.

There are several types of chronic MPN, based on the cells affected.Three classic types of MPN include polycythemia vera (PV), in whichthere are too many RBCs; essential thrombocythemia (ET), in which thereare too many platelets; primary myelofibrosis (PMF), in which fibers andblasts (abnormal stem cells) build up in the bone marrow. Other types ofMPN include: chronic myeloid leukemia, in which there are too many whiteblood cells; chronic neutrophilic leukemia, in which there are too manyneutrophils; chronic eosinophilic leukemia, not otherwise specified, inwhich there are too many eosinophils (hypereosinophilia); mastocytosis,also called mast cell disease, in which there are too many mast cells,which are a type of immune system cell found in tissues, like skin anddigestive organs, rather than in the bloodstream; myeloid and lymphoidneoplasms with eosinophilia and abnormalities of the PDGFRA, PDGFRB, andFGFR1 genes; and other unclassifiable myeloproliferative neoplasms.

In some embodiments, provided herein are methods to treat leukemia in asubject with an FTI or selecting leukemia patients for an FTI treatment.Leukemia refers to malignant neoplasms of the blood-forming tissues.Various forms of leukemias are described, for example, in U.S. Pat. No.7,393,862 and U.S. provisional patent application No. 60/380,842, filedMay 17, 2002, the entireties of which are incorporated herein byreference. Although viruses reportedly cause several forms of leukemiain animals, causes of leukemia in humans are to a large extent unknown.The Merck Manual, 944-952 (17^(th) ed. 1999). Transformation tomalignancy typically occurs in a single cell through two or more stepswith subsequent proliferation and clonal expansion. In some leukemias,specific chromosomal translocations have been identified with consistentleukemic cell morphology and special clinical features (e.g.,translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and17 in acute promyelocytic leukemia). Acute leukemias are predominantlyundifferentiated cell populations and chronic leukemias more mature cellforms.

Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. The Merck Manual, 946-949 (17^(th) ed.1999). They may be further subdivided by their morphologic andcytochemical appearance according to the French-American-British (FAB)classification or according to their type and degree of differentiation.The use of specific B- and T-cell and myeloid-antigen monoclonalantibodies are most helpful for classification. ALL is predominantly achildhood disease which is established by laboratory findings and bonemarrow examination. ANLL, also known as acute myelogenous leukemia orAML, occurs at all ages and is the more common acute leukemia amongadults; it is the form usually associated with irradiation as acausative agent. In some embodiments, provided herein are methods fortreating a AML patient with an FTI, or methods for selecting patientsfor FTI treatment.

Standard procedures treat AML patients usually include 2 chemotherapy(chemo) phases: remission induction (or induction) and consolidation(post-remission therapy). The first part of treatment (remissioninduction) is aimed at getting rid of as many leukemia cells aspossible. The intensity of the treatment can depend on a person's ageand health. Intensive chemotherapy is often given to people under theage of 60. Some older patients in good health can benefit from similaror slightly less intensive treatment. People who are much older or arein poor health are not suitable for intensive chemotherapies.

In younger patients, such as those under 60, induction often involvestreatment with 2 chemo drugs, cytarabine (ara-C) and an anthracyclinedrug such as daunorubicin (daunomycin) or idarubicin. Sometimes a thirddrug, cladribine (Leustatin, 2-CdA), is given as well. The chemo isusually given in the hospital and lasts about a week. In rare caseswhere the leukemia has spread to the brain or spinal cord, chemo mayalso be given into the cerebrospinal fluid (CSF). Radiation therapymight be used as well.

Induction is considered successful if remission is achieved. However,the AML in some patients can be refractory to induction. In patients whorespond to induction, further treatment is then given to try to destroyremaining leukemia cells and help prevent a relapse, which is calledconsolidation. For younger patients, the main options for consolidationtherapy are: several cycles of high-dose cytarabine (ara-C) chemo(sometimes known as HiDAC); allogeneic (donor) stem cell transplant; andautologous stem cell transplant.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic(CML). The Merck Manual, 949-952 (17^(th) ed. 1999). CLL ischaracterized by the appearance of mature lymphocytes in blood, bonemarrow, and lymphoid organs. The hallmark of CLL is sustained, absolutelymphocytosis (>5,000/μL) and an increase of lymphocytes in the bonemarrow. Most CLL patients also have clonal expansion of lymphocytes withB-cell characteristics. CLL is a disease of middle or old age. In CML,the characteristic feature is the predominance of granulocytic cells ofall stages of differentiation in blood, bone marrow, liver, spleen, andother organs. In the symptomatic patient at diagnosis, the total whiteblood cell (WBC) count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome. Bone marrow stromal cells are well knownto support CLL disease progression and resistance to chemotherapy.Disrupting the interactions between CLL cells and stromal cells is anadditional target of CLL chemotherapy.

Additionally, other forms of CLL include prolymphocytic leukemia (PLL),Large granular lymphocyte (LGL) leukemia, Hairy cell leukemia (HCL). Thecancer cells in PLL are similar to normal cells calledprolymphocytes—immature forms of B lymphocytes (B-PLL) or T lymphocytes(T-PLL). Both B-PLL and T-PLL tend to be more aggressive than the usualtype of CLL. The cancer cells of LGL are large and have features ofeither T cells or NK cells. Most LGL leukemias are slow-growing, but asmall number are more aggressive. HCL is another cancer of lymphocytesthat tends to progress slowly, and accounts for about 2% of allleukemias. The cancer cells are a type of B lymphocyte but are differentfrom those seen in CLL.

Juvenile myelomonocytic leukemia (JMML) is a serious chronic leukemiathat affects children mostly aged 4 and under. The average age ofpatients at diagnosis is 2 years old. The World Health Organization hascategorized JMML as a mixed myelodysplastic and myeloproliferativedisorder. The JMML encompasses diagnoses formerly referred to asJuvenile Chronic Myeloid Leukemia (JCML), Chronic MyelomonocyticLeukemia of Infancy, and Infantile Monosomy 7 Syndrome.

Lymphoma refers to cancers that originate in the lymphatic system.Lymphoma is characterized by malignant neoplasms of lymphocytes—Blymphocytes (B cell lymphoma), T lymphocytes (T-cell lymphoma), andnatural killer cells (NK cell lymphoma). Lymphoma generally starts inlymph nodes or collections of lymphatic tissue in organs including, butnot limited to, the stomach or intestines. Lymphoma may involve themarrow and the blood in some cases. Lymphoma may spread from one site toother parts of the body.

The treatments of various forms of lymphomas are described, for example,in U.S. Pat. No. 7,468,363, the entirety of which is incorporated hereinby reference. Such lymphomas include, but are not limited to, Hodgkin'slymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activatedB-cell lymphoma, Diffuse Large B-Cell Lymphoma (DLBCL), mantle celllymphoma (MCL), follicular lymphoma (FL; including but not limited to FLgrade I, FL grade II), follicular center lymphoma, transformed lymphoma,lymphocytic lymphoma of intermediate differentiation, intermediatelymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocyticlymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved celllymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Celllymphoma (CTCL) and mantle zone lymphoma and low grade follicularlymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common cancer for bothmen and women in the United States, with an estimated 63,190 new casesand 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007;57(1):43-66. The probability of developing NHL increases with age andthe incidence of NHL in the elderly has been steadily increasing in thepast decade, causing concern with the aging trend of the U.S.population. Id. Clarke C A, et al., Cancer 2002; 94(7):2015-2023.

DLBCL accounts for approximately one-third of non-Hodgkin's lymphomas.While some DLBCL patients are cured with traditional chemotherapy, theremainders die from the disease. Anticancer drugs cause rapid andpersistent depletion of lymphocytes, possibly by direct apoptosisinduction in mature T and B cells. See K. Stahnke. et al., Blood 2001,98:3066-3073. Absolute lymphocyte count (ALC) has been shown to be aprognostic factor in follicular non-Hodgkin's lymphoma and recentresults have suggested that ALC at diagnosis is an important prognosticfactor in DLBCL.

DLBCL can be divided into distinct molecular subtypes according to theirgene profiling patterns: germinal-center B-cell-like DLBCL (GCB-DLBCL),activated B-cell-like DLBCL (ABC-DLBCL), and primary mediastinal B-celllymphoma (PMBL) or unclassified type. These subtypes are characterizedby distinct differences in survival, chemo-responsiveness, and signalingpathway dependence, particularly the NF-κB pathway. See D. Kim et al.,Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings PartI. Vol 25, No. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al.,Blood 2005; 106: 3183-90; Ngo V. N. et al., Nature 2011; 470: 115-9.Such differences have prompted the search for more effective andsubtype-specific treatment strategies in DLBCL. In addition to the acuteand chronic categorization, neoplasms are also categorized based uponthe cells giving rise to such disorder into precursor or peripheral. Seee.g., U.S. patent Publication No. 2008/0051379, the disclosure of whichis incorporated herein by reference in its entirety. Precursor neoplasmsinclude ALLs and lymphoblastic lymphomas and occur in lymphocytes beforethey have differentiated into either a T- or B-cell. Peripheralneoplasms are those that occur in lymphocytes that have differentiatedinto either T- or B-cells. Such peripheral neoplasms include, but arenot limited to, B-cell CLL, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma,extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoidtissue, nodal marginal zone lymphoma, splenic marginal zone lymphoma,hairy cell leukemia, plasmacytoma, Diffuse large B-cell lymphoma (DLBCL)and Burkitt lymphoma. In over 95 percent of CLL cases, the clonalexpansion is of a B cell lineage. See Cancer: Principles & Practice ofOncology (3rd Edition) (1989) (pp. 1843-1847). In less than 5 percent ofCLL cases, the tumor cells have a T-cell phenotype. Notwithstandingthese classifications, however, the pathological impairment of normalhematopoiesis is the hallmark of all leukemias.

PTCL consists of a group of rare and usually aggressive (fast-growing)NHLs that develop from mature T-cells. PTCLs collectively account forabout 4 to 10 percent of all NHL cases, corresponding to an annualincidence of 2,800-7,200 patients per year in the United States. By someestimates, the incidence of PTCL is growing significantly, and theincreasing incidence may be driven by an aging population. PTCLs aresub-classified into various subtypes, each of which are typicallyconsidered to be separate diseases based on their distinct clinicaldifferences. Most of these subtypes are rare; the three most commonsubtypes of PTCL not otherwise specified, anaplastic large-celllymphoma, or ALCL, and angioimmunoblastic T-cell lymphoma, thatcollectively account for approximately 70 percent of all PTCLs in theUnited States. ALCL can be cutaneous ALCL or systemic ALCL.

For most PTCL subtypes, the frontline treatment regimen is typicallycombination chemotherapy, such as CHOP (cyclophosphamide, doxorubicin,vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin,cyclophosphamide, prednisone), or other multi-drug regimens. Patientswho relapse or are refractory to frontline treatments are typicallytreated with gemcitabine in combination with other chemotherapies,including vinorelbine (Navelbine®) and doxorubicin (Doxil®) in a regimencalled GND, or other chemotherapy regimens such as DHAP (dexamethasone,cytarabine, cisplatin) or ESHAP (etoposide, methylprednisolone,cytarabine, and cisplatin).

Because most patients with PTCL will relapse, some oncologists recommendgiving high-dose chemotherapy followed by an autologous stem celltransplant to some patients who had a good response to their initialchemotherapy. Recent, non-cytotoxic therapies that have been approvedfor relapsed or refractory PTCL, such as pralatrexate (Folotyn®),romidepsin (Istodax®) and belinostat (Beleodaq®), are associated withrelatively low objective response rates (25-27% overall response rate,or ORR) and relatively short durations of response (8.2-9.4 months).Accordingly, the treatment of relapsed/refractory PTCL remains asignificant unmet medical need.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow.Normally, plasma cells produce antibodies and play a key role in immunefunction. However, uncontrolled growth of these cells leads to bone painand fractures, anemia, infections, and other complications. Multiplemyeloma is the second most common hematological malignancy, although theexact causes of multiple myeloma remain unknown. Multiple myeloma causeshigh levels of proteins in the blood, urine, and organs, including butnot limited to M-protein and other immunoglobulins (antibodies),albumin, and beta-2-microglobulin. M-protein, short for monoclonalprotein, also known as paraprotein, is a particularly abnormal proteinproduced by the myeloma plasma cells and can be found in the blood orurine of almost all patients with multiple myeloma.

Skeletal symptoms, including bone pain, are among the most clinicallysignificant symptoms of multiple myeloma. Malignant plasma cells releaseosteoclast stimulating factors (including IL-1, IL-6 and TNF) whichcause calcium to be leached from bones causing lytic lesions;hypercalcemia is another symptom. The osteoclast stimulating factors,also referred to as cytokines, may prevent apoptosis, or death ofmyeloma cells. Fifty percent of patients have radiologically detectablemyeloma-related skeletal lesions at diagnosis. Other common clinicalsymptoms for multiple myeloma include polyneuropathy, anemia,hyperviscosity, infections, and renal insufficiency.

Bone marrow stromal cells are well known to support multiple myelomadisease progression and resistance to chemotherapy. Disrupting theinteractions between multiple myeloma cells and stromal cells is anadditional target of multiple myeloma chemotherapy.

In some embodiments, provided herein are methods for predictingresponsiveness of a MDS patient to an FTI treatment, methods for MDSpatient population selection for an FTI treatment, and methods fortreating MDS in a subject with a therapeutically effective amount of anFTI, based on the mutation status of Ras in a sample from the patient.In some embodiments, provided herein are methods for predictingresponsiveness of a MPN patient to an FTI treatment, methods for MDSpatient population selection for an FTI treatment, and methods fortreating MPN in a subject with a therapeutically effective amount of anFTI, based on the mutation status of Ras in a sample from the patient.In some embodiments, provided herein are methods for predictingresponsiveness of a AML patient to an FTI treatment, methods for AMLpatient population selection for an FTI treatment, and methods fortreating AML in a subject with a therapeutically effective amount of anFTI, based on the mutation status of Ras in a sample from the patient.In some embodiments, provided herein are methods for predictingresponsiveness of a JMML patient to an FTI treatment, methods for JMMLpatient population selection for an FTI treatment, and methods fortreating JMML in a subject with a therapeutically effective amount of anFTI, based on the mutation status of Ras in a sample from the patient.

In some embodiments, provided herein are methods for predictingresponsiveness of a CMML patient to an FTI treatment, methods for CMMLpatient population selection for an FTI treatment, and methods fortreating CMML in a subject with a therapeutically effective amount of anFTI, based on the mutation status of Ras in a sample from the patient.In some embodiments, provided herein is a method of treating CMML in asubject based on the mutation status of K-Ras, N-Ras, or both. Themethod provided herein includes (a) determining the presence or absenceof a Ras mutation in a sample from the subject, wherein the Ras mutationincludes a K-Ras mutation or a N-Ras mutation, and subsequently (b)administering a therapeutically effective amount of an FTI to saidsubject if said sample is determined to lack the K-Ras mutation or theN-Ras mutation. In some embodiments, the sample is determined to nothave any K-Ras mutation or any N-Ras mutation. In some embodiments, thesample is determined to have wild type K-Ras. In some embodiments, thesample is determined to have wild type N-Ras. In some embodiments, thesample is determined to have wild type K-Ras and wild type N-Ras. Insome embodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods for predictingresponsiveness of a CMML patient to tipifarnib, methods for CMML patientpopulation selection for tipifarnib treatment, and methods for treatingCMML in a subject with a therapeutically effective amount of antipifarnib, based on the mutation status of K-Ras and N-Ras in a samplefrom the patient. In some embodiments, provided herein is a method oftreating CMML in a subject based on the mutation status of K-Ras, N-Ras,or both. The method provided herein includes (a) determining thepresence or absence of a Ras mutation in a sample from a subject havingCMML, wherein the Ras mutation includes a K-Ras mutation or a N-Rasmutation, and subsequently (b) administering a therapeutically effectiveamount of tipifarnib to the subject if the sample is determined to lackthe K-Ras mutation or the N-Ras mutation. In some embodiments, thesample is determined to not have any K-Ras mutation or any N-Rasmutation. In some embodiments, the sample is determined to have wildtype K-Ras. In some embodiments, the sample is determined to have wildtype N-Ras. In some embodiments, the sample is determined to have wildtype K-Ras and wild type N-Ras.

4.4. EXEMPLARY FTIS AND DOSAGES

In some embodiments, provided herein is a method of treating a cancer ina subject based on the mutation status of K-Ras, N-Ras, or both. Themethod provided herein includes (a) determining the presence or absenceof a Ras mutation in a sample from the subject, wherein the Ras mutationincludes a K-Ras mutation or a N-Ras mutation, and subsequently (b)administering a therapeutically effective amount of tipifarnib to saidsubject if said sample is determined to lack the K-Ras mutation or theN-Ras mutation. In some embodiments, the sample is determined to havewild type K-Ras. In some embodiments, the sample is determined to havewild type N-Ras. In some embodiments, the sample is determined to havewild type K-Ras and wild type N-Ras. In some embodiments, the methodsinclude administering the subject with another FTI described herein orotherwise known in the art. In some embodiments, the FTI is selectedfrom the group consisting of tipifarnib, arglabin, perrilyl alcohol,lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754,R208176, AZD3409, and BMS-214662.

In some embodiments, provided herein is a method of treating ahematological cancer in a subject based on the mutation status of K-Ras,N-Ras, or both. The method provided herein includes (a) determining thepresence or absence of a Ras mutation in a sample from the subject,wherein the Ras mutation includes a K-Ras mutation or a N-Ras mutation,and subsequently (b) administering a therapeutically effective amount oftipifarnib to said subject if said sample is determined to lack theK-Ras mutation or the N-Ras mutation. In some embodiments, the sample isdetermined to have wild type K-Ras. In some embodiments, the sample isdetermined to have wild type N-Ras. In some embodiments, the sample isdetermined to have wild type K-Ras and wild type N-Ras. In someembodiments, the methods include administering the subject with anotherFTI described herein or otherwise known in the art. In some embodiments,the FTI is selected from the group consisting of tipifarnib, arglabin,perrilyl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277,L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, provided herein is a method of treating CMML in asubject based on the mutation status of K-Ras, N-Ras, or both. Themethod provided herein includes (a) determining the presence or absenceof a Ras mutation in a sample from the subject, wherein the Ras mutationincludes a K-Ras mutation or a N-Ras mutation, and subsequently (b)administering a therapeutically effective amount of tipifarnib to saidsubject if said sample is determined to lack the K-Ras mutation or theN-Ras mutation. In some embodiments, the sample is determined to havewild type K-Ras. In some embodiments, the sample is determined to havewild type N-Ras. In some embodiments, the sample is determined to havewild type K-Ras and wild type N-Ras. In some embodiments, the methodsinclude administering the subject with another FTI described herein orotherwise known in the art. In some embodiments, the FTI is selectedfrom the group consisting of tipifarnib, arglabin, perrilyl alcohol,lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754,R208176, AZD3409, and BMS-214662.

In some embodiments, the FTI is administered orally, parenterally,rectally, or topically. In some embodiments, the FTI is administeredorally. In some embodiments, tipifarnib is administered orally,parenterally, rectally, or topically. In some embodiments, tipifarnib isadministered orally.

In some embodiments, the FTI is administered at a dose of 1-1000 mg/kgbody weight. In some embodiments, the FTI is administered twice a day.In some embodiments, the FTI is administered at a dose of 200-1200 mgtwice a day. In some embodiments, the FTI is administered at a dose of600 mg twice a day. In some embodiments, the FTI is administered at adose of 900 mg twice a day. In some embodiments, tipifarnib isadministered at a dose of 1-1000 mg/kg body weight. In some embodiments,tipifarnib is administered twice a day. In some embodiments, tipifarnibis administered at a dose of 200-1200 mg twice a day. In someembodiments, tipifarnib is administered at a dose of 600 mg twice a day.In some embodiments, tipifarnib is administered at a dose of 900 mgtwice a day.

In some embodiments, the FTI is administered at a dose of 1-1000 mg/kgbody weight. In some embodiments, the FTI is administered twice a day.In some embodiments, the FTI is administered at a dose of 200-1200 mgtwice a day. In some embodiments, the FTI is administered at a dose of600 mg twice a day. In some embodiments, the FTI is administered at adose of 900 mg twice a day. In some embodiments, tipifarnib isadministered in treatment cycles. In some embodiments, tipifarnib isadministered in alternative weeks. In some embodiments, tipifarnib isadministered on days 1-7 and 15-21 of a 28-day treatment cycle. In someembodiments, tipifarnib is administered orally at a dose of 900 mg twicea day on days 1-7 and 15-21 of a 28-day treatment cycle.

In some embodiments, the FTI is administered for at least 3 cycles. Insome embodiments, the FTI is administered for at least 6 cycles. In someembodiments, the FTI is administered for up to 12 cycles. In someembodiments, the FTI is administered orally at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle for at least threecycles. In some embodiments, tipifarnib is administered for at least 3cycles. In some embodiments, tipifarnib is administered for at least 6cycles. In some embodiments, tipifarnib is administered for up to 12cycles. In some embodiments, tipifarnib is administered orally at a doseof 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatment cyclefor at least three cycles.

In some embodiments, provided herein are methods for treating CMML in asubject with a therapeutically effective amount of an tipifarnib, basedon the mutation status of K-Ras in a sample from the patient. In someembodiments, provided herein is a method of treating CMML in a subjectincluding (a) determining a sample from the subject to have wild typeK-Ras, and subsequently (b) administering tipifarnib to the subject at adose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle.

In some embodiments, provided herein are methods for treating CMML in asubject with a therapeutically effective amount of an tipifarnib, basedon the mutation status of N-Ras in a sample from the patient. In someembodiments, provided herein is a method of treating CMML in a subjectincluding (a) determining a sample from the subject to have wild typeN-Ras, and subsequently (b) administering tipifarnib to the subject at adose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle.

In some embodiments, provided herein are methods for treating CMML in asubject with a therapeutically effective amount of an tipifarnib, basedon the mutation status of K-Ras and N-Ras in a sample from the patient.In some embodiments, provided herein is a method of treating CMML in asubject including (a) determining a sample from the subject to have wildtype K-Ras and wild type N-Ras, and subsequently (b) administeringtipifarnib to the subject at a dose of 900 mg twice a day on days 1-7and 15-21 of a 28-day treatment cycle.

5. MUTANT H-RAS AS BIOMARKERS FOR FTI TREATMENT 5.1. H-RAS MUTATIONSTATUS

The H-ras protein is involved in regulating cell division in response togrowth factor stimulation. Growth factors act by binding cell surfacereceptors that span the cell's plasma membrane. Once activated,receptors stimulate signal transduction events in the cytoplasm, aprocess by which proteins and second messengers relay signals fromoutside the cell to the cell nucleus and instruct the cell to grow ordivide. H-ras is localized in the plasma membrane, and is an earlyplayer in many signal transduction pathways. H-ras acts as a molecularon/off switch—once it is turned on it recruits and activates proteinsnecessary for the propagation of the receptor's signal. In certaintumors, mutations in H-ras or its upstream effectors cause it to bepermanently on, resulting in persistent activation of downstream growthand proliferation signals that drive tumor cell growth. FTIs work toprevent the aberrant growth and proliferation of cells that aredependent on these signaling pathways by inhibiting proteinfarnesylation and subsequent membrane localization of H-ras, therebyswitching H-ras off.

FTIs such as tipifarnib prevent protein farnesylation, a type of proteinmodification known as prenylation, which along with other proteinmodifications, allows membrane localization of H-ras where it canreceive and transmit extracellular signals implicated in cancerinitiation and development. FTIs such as tipifarnib can block H-rasfarnesylation and subsequent membrane localization, and inhibitoncogenic, H-ras-driven cellular transformation in vitro and in vivo.While K-ras and N-ras similarly utilize protein farnesylation, they canalso utilize a related prenylation pathway that also leads to membranelocalization. Meanwhile, H-ras membrane localization is solely dependenton protein farnesylation.

In some embodiments, the cancer to be treated by methods provided hereincan have H-ras mutations. In some embodiments, the cancer to be treatedby methods provided herein can be a solid tumor with a H-ras mutation.The solid tumor with H-ras mutation can be any of the solid tumordescribed above. In some embodiments, the solid tumor can be thyroidcancers, head and neck cancers, urothelial carcinomas, bladder cancersor salivary cancers with H-ras mutation. Methods provided herein orotherwise known in the art can be used to determine the mutation statusof a ras gene. In some embodiments, the mutation status of a ras genecan be determined an NGS-based assay. In some embodiments, the mutationstatus of a ras gene can be determined by a qualitative PCR-based assay.A qualitative PCR based assay can be technically similar to thePCR-based assays already developed and approved by the FDA for K-ras. Insome embodiments, mutation status of a ras gene can be determined in theform of a companion diagnostic to the FTI treatment, such as thetipifarnib treatment. The companion diagnostic can be performed at theclinic site where the patient receives the tipifarnib treatment, or at aseparate site.

Provided herein are methods of selection of cancer patients fortreatment with an FTI based on the presence of a H-Ras mutation. Thesemethods are based, in part, on the discovery that H-Ras mutation isassociated with clinical benefits of FTI treatment, and thus can be usedto predict the responsiveness of a cancer patient to an FTI treatment.Accordingly, provided herein are methods for predicting responsivenessof a cancer patient to an FTI treatment, methods for cancer patientpopulation selection for an FTI treatment, and methods for treatingcancer in a subject with a therapeutically effective amount of an FTI,based on the presence of H-Ras mutation in a sample from the patient.The cancer can be a hematopoietic cancer or a solid tumor. In someembodiments, the cancer is a solid tumor.

In some embodiments, provided herein is a method of treating a cancer ina subject based on the presence of a H-Ras mutation. The method providedherein includes (a) determining the presence or absence of a H-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a H-Ras mutation. The samplecan be a tumor sample. In some embodiments, the methods include (a)determining a cancer patient to have a H-Ras mutation, and subsequently(b) administering a therapeutically effective amount of an FTI to thesubject.

In some embodiments, the H-Ras mutation is a mutation at a codonselected from the group consisting of G12, G13, and Q61. In someembodiments, the H-Ras mutation can be a mutation selected from thegroup consisting of the amino acid substitutions of G12R, G12V, G13C,G13R, Q61L and Q61R. In some embodiments, the mutation can be mutationat other codon that result in activation of H-Ras protein.

In some embodiments, the methods provided herein further include (a)determining the presence or absence of a K-Ras mutation and a N-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample does not have the K-Ras mutation or the N-Rasmutation. In some embodiment, the method includes administering atherapeutically effective amount of an FTI to the subject if the samplehas wild type K-Ras and wild type N-Ras.

In some embodiments, the K-Ras mutation is K_(A)-Ras mutation. In someembodiments, the K-Ras mutation is K_(B)-Ras mutation. In someembodiments, the K-Ras mutation is a combination of K_(A)-Ras mutationand a K_(B)-Ras mutation. The K-Ras mutation can include a mutation at acodon selected from the group consisting of G12, G13, and Q61 ofK_(A)-Ras, K_(B)-Ras, or both. In some embodiments, the K_(A)-Rasmutation can include a mutation selected from the group consisting ofthe amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R,G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R andA146V. In some embodiments, the K_(B)-Ras mutation can include amutation selected from the group consisting of the amino acidsubstitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C,G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the N-Ras mutation can include at least onemutation at a codon selected from the group consisting of G12, G13, G15,G60 and Q61. In some embodiments, the N-Ras mutation can include atleast one mutation at a codon selected from the group consisting of G12,G13, and Q61. In some embodiments, the N-Ras mutation can include atleast one mutation selected from the group consisting of the amino acidsubstitutions of G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R,G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the sample is determined to not have amino acidsubstitution at G12, G13, and Q61 of K-Ras, and also not have amino acidsubstitution at G12, G13, and Q61 of N-Ras. In some embodiments, thesample is determined to not have any K-Ras mutation or any N-Rasmutation. In some embodiments, the sample is determined to have wildtype K-Ras and wild type N-Ras.

In some embodiments, the method provided herein includes (a) determiningthe presence or absence of a H-Ras mutation, a K-Ras mutation, and aN-Ras mutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a H-Ras mutation, but noK-Ras mutation or N-Ras mutation. The sample can be a tumor sample. Insome embodiments, the methods include (a) determining a cancer patientto have a H-Ras mutation and wild type K-Ras and wild type N-Ras, andsubsequently (b) administering a therapeutically effective amount of anFTI to the subject. In some embodiments, the FTI is tipifarnib.

Provided herein are methods to treat a cancer in a subject with an FTI,and methods for selecting cancer patients for an FTI treatment based onthe presence of a H-Ras mutation. The cancer can be a hematopoieticcancer or a solid tumor. Provided herein are also methods to treat apremalignant condition in a subject with an FTI, and methods forselecting patients with a premalignant condition for an FTI treatmentbased on H-Ras mutation status.

In some embodiments, provided herein are methods to treat a cancer in asubject with an FTI or selecting cancer patients for an FTI treatmentbased on the presence of a H-Ras mutation. The cancer can be ahematopoietic cancer or a solid tumor. The cancer can be related toHuman papillomavirus (HPV+ or HPV positive), or unrelated to HPV (HPV−or HPV negative).

Provided herein are methods for predicting responsiveness of a cancerpatient to an FTI treatment, methods for cancer patient populationselection for an FTI treatment, and methods for treating cancer in asubject with a therapeutically effective amount of an FTI, based on thepresence of a H-Ras mutation in a sample from the patient. The mutationstatus of H-Ras can be detected at the nucleic acid or protein level. Insome embodiments, the H-Ras mutation status is determined by analyzingnucleic acids obtained from the sample. In some embodiments, the H-Rasmutation status is determined by analyzing protein obtained from thesample.

In some embodiments, the H-Ras mutation status is determined byanalyzing nucleic acids obtained from the sample. The nucleic acids maybe mRNA or genomic DNA molecules from the test subject. Methods fordetermining Ras mutation status by analyzing nucleic acids are wellknown in the art. In some embodiments, the methods include sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) assay, denaturing high-performanceliquid chromatography (DHPLC), or Restriction Fragment LengthPolymorphism (RFLP) assay. In some embodiments, the Ras mutation statusis determined using standard sequencing methods, including, for example,Sanger sequencing, next generation sequencing (NGS). In someembodiments, the Ras mutation status is determined using MS.

In some embodiments, the H-Ras mutation status is determined byanalyzing protein obtained from the sample. The mutated Ras H-proteincan be detected by a variety of immunohistochemistry (IHC) approaches,Immunoblotting assay, Enzyme-Linked Immunosorbent Assay (ELISA) or otherimmunoassay methods known in the art.

As a person of ordinary skill in the art would understand, any methodsdescribed herein or otherwise known in the art for analyzing Rasmutation can be used to determining the presence or absence of a H-Rasmutation.

5.2. SAMPLES

In some embodiments, methods provided herein include obtaining a samplefrom the subject. In some embodiments, the sample is a tumor sample. Insome embodiments, the sample used in the present methods includes abiopsy (e.g., a tumor biopsy). The biopsy can be from any organ ortissue, for example, skin, liver, lung, heart, colon, kidney, bonemarrow, teeth, lymph node, hair, spleen, brain, breast, or other organs.Any biopsy technique known by those skilled in the art can be used forisolating a sample from a subject, for instance, open biopsy, closebiopsy, core biopsy, incisional biopsy, excisional biopsy, or fineneedle aspiration biopsy.

The sample used in the methods provided herein includes body fluids froma subject. Non-limiting examples of body fluids include blood (e.g.,peripheral whole blood, peripheral blood), blood plasma, bone marrow,amniotic fluid, aqueous humor, bile, lymph, menses, serum, urine,cerebrospinal fluid surrounding the brain and the spinal cord, synovialfluid surrounding bone joints.

In one embodiment, the sample is a bone marrow sample. Procedures toobtain a bone marrow sample are well known in the art, including but notlimited to bone marrow biopsy and bone marrow aspiration. Bone marrowhas a fluid portion and a more solid portion. In bone marrow biopsy, asample of the solid portion is taken. In bone marrow aspiration, asample of the fluid portion is taken. Bone marrow biopsy and bone marrowaspiration can be done at the same time and referred to as a bone marrowexam.

In some embodiments, the sample is a blood sample. The blood sample canbe obtained using conventional techniques as described in, e.g. Innis etal, editors, PCR Protocols (Academic Press, 1990). White blood cells canbe separated from blood samples using convention techniques orcommercially available kits, e.g. RosetteSep kit (Stein CellTechnologies, Vancouver, Canada). Sub-populations of white blood cells,e.g. mononuclear cells, NK cells, B cells, T cells, monocytes,granulocytes or lymphocytes, can be further isolated using conventionaltechniques, e.g. magnetically activated cell sorting (MACS) (MiltenyiBiotec, Auburn, Calif.) or fluorescently activated cell sorting (FACS)(Becton Dickinson, San Jose, Calif.).

In certain embodiments, the sample used in the methods provided hereinincludes a plurality of cells. Such cells can include any type of cells,e.g., stem cells, blood cells (e.g., PBMCs), lymphocytes, NK cells, Bcells, T cells, monocytes, granulocytes, immune cells, or tumor orcancer cells. Specific cell populations can be obtained using acombination of commercially available antibodies (e.g., Quest Diagnostic(San Juan Capistrano, Calif.); Dako (Denmark)).

In certain embodiments, the sample used in the methods provided hereinis from a diseased tissue, e.g., from an individual having cancer (e.g.a head and neck cancer, a salivary gland tumor, or a thyroid tumor). Incertain embodiments. In some embodiments, the cells can be obtained fromthe tumor or cancer cells or a tumor tissue, such as a tumor biopsy or atumor explants. In certain embodiments, the number of cells used in themethods provided herein can range from a single cell to about 10⁹ cells.In some embodiments, the number of cells used in the methods providedherein is about 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,1×10⁸, or 5×10⁸.

The number and type of cells collected from a subject can be monitored,for example, by measuring changes in morphology and cell surface markersusing standard cell detection techniques such as flow cytometry, cellsorting, immunocytochemistry (e.g., staining with tissue specific orcell-marker specific antibodies) fluorescence activated cell sorting(FACS), magnetic activated cell sorting (MACS), by examination of themorphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. Fluorescence activated cell sorting (FACS) is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In certain embodiments, subsets of cells are used in the methodsprovided herein. Methods to sort and isolate specific populations ofcells are well-known in the art and can be based on cell size,morphology, or intracellular or extracellular markers. Such methodsinclude, but are not limited to, flow cytometry, flow sorting, FACS,bead based separation such as magnetic cell sorting, size-basedseparation (e.g., a sieve, an array of obstacles, or a filter), sortingin a microfluidics device, antibody-based separation, sedimentation,affinity adsorption, affinity extraction, density gradientcentrifugation, laser capture microdissection, etc.

5.3. CANCERS

In some embodiments, provided herein are methods to treat ahematopoietic cancer in a subject with an FTI or selecting cancerpatients for an FTI treatment based on the presence of a H-Ras mutation.In some embodiments, the hematopoietic cancer is HPV negative. In someembodiments, the methods include (a) determining a HPV negativehematopoietic cancer patient to have a H-Ras mutation, and subsequently(b) administering a therapeutically effective amount of tipifarnib tothe patient.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include myeloproliferativeneoplasm (MPN), myelodysplastic syndrome (MDS), leukemia, and lymphoma.In some embodiments, the cancer is acute myeloid leukemia (AML), naturalkiller cell lymphoma (NK lymphoma), natural killer cell leukemia (NKleukemia), cutaneous T-Cell lymphoma (CTCL), juvenile myelomonocyticleukemia (JMML), peripheral T-cell lymphoma (PTCL), chronic myeloidleukemia (CML) or chronic myelomonocytic leukemia (CMML). In someembodiments, the cancer is CMML. In some embodiments, the cancer isJMML.

In some embodiments, provided herein are methods to treat a solid tumorwith an FTI based on the presence of a H-Ras mutation. In someembodiments, the solid tumor is HPV negative. In some embodiments, theFTI is tipifarnib. In some embodiments, the methods include (a)determining a HPV negative solid tumor patient to have a H-Ras mutation,and subsequently (b) administering a therapeutically effective amount oftipifarnib to the patient.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). The solid tumorto be treated with the methods of the invention can be sarcomas andcarcinomas, include head and neck carcinoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, thyroid carcinoma, medullarythyroid carcinoma, papillary thyroid carcinoma, pheochromocytomassebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer, testicular tumor, seminoma, bladder carcinoma,melanoma, and CNS tumors (such as a glioma (such as brainstem glioma andmixed gliomas), glioblastoma (also known as glioblastoma multiforme)astrocytoma, CNS lymphoma, germinoma, meduloblastoma, Schwannomacraniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastomaand brain metastases).

In some embodiments, provided herein are methods to treat a solid tumorwith an FTI based on the presence of a H-Ras mutation, wherein the solidtumor is thyroid cancer, head and neck cancers, salivary gland cancers,malignant melanoma, adrenal carcinoma, breast carcinoma, renal cellcancer, carcinoma of the pancreas, non-small-cell lung carcinoma (NSCLC)or carcinoma of unknown primary. In some embodiments, the FTI istipifarnib. Drugs commonly administered to patients with various typesor stages of solid tumors include, but are not limited to, celebrex,etoposide, cyclophosphamide, docetaxel, apecitabine, IFN, tamoxifen,IL-2, GM-CSF, or a combination thereof.

In some embodiments, the solid tumor to be treated by methods providedherein can be thyroid cancer, head and neck cancers, urothelial cancers,salivary cancers, cancers of the upper digestive tract, bladder cancer,breast cancer, ovarian cancer, brain cancer, gastric cancer, prostatecancer, lung cancer, colon cancer, skin cancer, liver cancer, andpancreatic cancer. In some embodiments, the bladder cancer to be treatedby methods provided herein can be transitional cell carcinoma. In someembodiments, the FTI is tipifarnib.

In some embodiments, the solid tumor to be treated by methods providedherein can be selected from the groups consisting of carcinoma,melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the premalignant conditions to be treated bymethods provided herein can be actinic cheilitis, Barrett's esophagus,atrophic gastritis, ductal carcinoma in situ, Dyskeratosis congenita,Sideropenic dysphagia, Lichen planus, Oral submucous fibrosis, Solarelastosis, cervical dysplasia, polyps, leukoplakia, erythroplakia,squamous intraepithelial lesion, a pre-malignant disorder, or apre-malignant immunoproliferative disorder.

In some embodiments, provided herein are methods to treat a solid tumorin a subject with an FTI and methods to select a solid tumor patientsfor FTI treatment based on the presence of a H-Ras mutation in thesubject, wherein the solid tumor is thyroid cancer, head and neckcancers, or salivary gland cancer. In some embodiments, the solid tumoris thyroid cancer. In some embodiments, the solid tumor is head and necksquamous cell carcinoma (HNSCC). In some embodiments, the solid tumor issalivary gland cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6^(th) most commoncancer worldwide, with about 650,000 cases and 200,000 deaths per yearworldwide, and about 54,000 new cases per year in the US. It is also themost common cancer in central Asia.

HNSCC has 2 different etiologies and corresponding tumor types. Thefirst subtype is associated with tobacco smoking and alcoholconsumption, and unrelated to Human papillomavirus (HPV− or HPVnegative). The second subtype is associated with infection withhigh-risk HPV (HPV+ or HPV positive). The second subtype is largelylimited to oropharyngeal cancers. HPV+ tumors are distinct entity withbetter prognosis and may require differential treatments.

Significant proportion of HNSCC, particularly oropharyngeal cancers, arecaused by HPV infection. High-risk HPV subtype 16 accounts for 85% ofall HPV+ tumors in HNSCC. P16 can be used as surrogate marker of HPVinfection in HNSCC, particularly in the oropharynx. More accurate HPVtesting is available and based on E6/E7 detection (Liang C, et al.Cancer Res. 2012; 72:5004-5013).

HPV+ HNSCC show significantly lower EGFR expression levels than HPV−HNSCC. EGFR amplification only occurs in HPV− HNSCC. High EGFR gene copynumber and protein expression are associated with poor clinical outcomein advanced HNSCC.

Currently, first-line therapy for recurrent/metastatic HNSCC includeplatinum-based doublet (e.g., cisplatin/5-FU or carboplatin/paclitaxel),optionally in combination with anti-EGFR antibody therapy (e.g.Cetuximab, Panitumumab, Afatinib). Second-line therapy includes taxanes,methotrexate, and/or cetuximab. Anti-EGFR antibody therapy, such asCetuximab (a chimeric IgG1) or Panitumumab can be used as a singleagent, with chemotherapy (e.g. Platinum/5-FU, Cisplatin), or withradiation therapy. Despite high EGFR expression levels in HNSCC,single-agent response rate for Cetuximab is only 13% with SD rate of33%, and there is currently no predictive biomarker available.

Drugs in development for HNSCC include those targeting PI3K pathway:BKM120 (buparlisib)+cetuximab, BYL719+cetuximab, Temsirolimus+cetuximab,Rigosertib+cetuximab; those targeting MET pathway: Tivantinib+cetuximab,Ficlatuzumab+cetuximab; those targeting EGFR/HER3 pathwayAfatinib+cetuximab±paclitaxel, Patritumab; those targeting FGFR pathway:BGJ398; those targeting CDK4/6-cell cycle pathway: Palbociclib, LEE011;RTK inhibitor: Anlotinib and chemotherapy: Oral Azacitidine. More recenttherapeutic options for HNSCC include immunotherapy, such as anti-PD1 oranti-PDL1 antibodies.

While high cure rates have been achieved for localized and loco-regionaldisease using surgery, radiation, chemoradiation, and inductionchemotherapy, survival rates for recurrent/metastatic diseases remainvery poor, and better treatment options are necessary.

In some embodiments, provided herein is a method of treating a HNSCC ina subject based on the presence of a H-Ras mutation. In someembodiments, the HNSCC can be HPV negative HNSCC. In some embodiments,the HNSCC can be relapsed/recurrent HNSCC. In some embodiments, theHNSCC can be metastatic HNSCC. The method provided herein includes (a)determining the presence or absence of a H-Ras mutation in a sample fromthe subject, and subsequently (b) administering a therapeuticallyeffective amount of an FTI to the subject if the sample is determined tohave a H-Ras mutation. The sample can be a tumor sample. In someembodiments, the methods include (a) determining a HNSCC patient to havea H-Ras mutation, and subsequently (b) administering a therapeuticallyeffective amount of an FTI to the subject. In some embodiments, the FTIis tipifarnib.

In some embodiments, provided herein is a method of treating a salivarygland cancer in a subject based on the presence of a H-Ras mutation. Insome embodiments, the salivary gland cancer can be advanced salivarygland cancer. In some embodiments, the salivary gland cancer can bemetastatic salivary gland cancer. The method provided herein includes(a) determining the presence or absence of a H-Ras mutation in a samplefrom the subject, and subsequently (b) administering a therapeuticallyeffective amount of an FTI to the subject if the sample is determined tohave a H-Ras mutation. The sample can be a tumor sample. In someembodiments, the methods include (a) determining a salivary gland cancerpatient to have a H-Ras mutation, and subsequently (b) administering atherapeutically effective amount of an FTI to the subject. In someembodiments, the FTI is tipifarnib.

In some embodiments, provided herein is a method of treating a thyroidcancer in a subject based on the presence of a H-Ras mutation. In someembodiments, the thyroid cancer can be relapsed/recurrent thyroidcancer. In some embodiments, the thyroid cancer can be metastaticthyroid cancer. In some embodiments, the thyroid cancer can be advancedthyroid cancer. The method provided herein includes (a) determining thepresence or absence of a H-Ras mutation in a sample from the subject,and subsequently (b) administering a therapeutically effective amount ofan FTI to the subject if the sample is determined to have a H-Rasmutation. The sample can be a tumor sample. In some embodiments, themethods include (a) determining a HNSCC patient to have a H-Rasmutation, and subsequently (b) administering a therapeutically effectiveamount of an FTI to the subject. In some embodiments, the FTI istipifarnib.

5.4. EXEMPLARY FTIS AND DOSAGES

In some embodiments, provided herein are methods to treat a cancer in asubject with an tipifarnib or selecting cancer patients for tipifarnibtreatment based on the presence of a H-Ras mutation. In someembodiments, the methods include treating the subject with another FTIdescribed herein or otherwise known in the art. In some embodiments, theFTI is selected from the group consisting of tipifarnib, arglabin,perrilyl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277,L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the FTI is administered orally, parenterally,rectally, or topically. In some embodiments, the FTI is administeredorally. In some embodiments, tipifarnib is administered orally,parenterally, rectally, or topically. In some embodiments, tipifarnib isadministered orally.

In some embodiments, the FTI is administered at a dose of 1-1000 mg/kgbody weight. In some embodiments, the FTI is administered twice a day.In some embodiments, the FTI is administered at a dose of 200-1200 mgtwice a day. In some embodiments, the FTI is administered at a dose of600 mg twice a day. In some embodiments, the FTI is administered at adose of 900 mg twice a day. In some embodiments, tipifarnib isadministered at a dose of 1-1000 mg/kg body weight. In some embodiments,tipifarnib is administered twice a day. In some embodiments, tipifarnibis administered at a dose of 200-1200 mg twice a day. In someembodiments, tipifarnib is administered at a dose of 600 mg twice a day.In some embodiments, tipifarnib is administered at a dose of 900 mgtwice a day.

In some embodiments, the FTI is administered in treatment cycles. Insome embodiments, the FTI is administered in alternative weeks. In someembodiments, the FTI is administered on days 1-7 and 15-21 of a 28-daytreatment cycle. In some embodiments, the FTI is administered orally ata dose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle. In some embodiments, tipifarnib is administered in treatmentcycles. In some embodiments, tipifarnib is administered in alternativeweeks. In some embodiments, tipifarnib is administered on days 1-7 and15-21 of a 28-day treatment cycle. In some embodiments, tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle.

In some embodiments, the FTI is administered for at least 3 cycles. Insome embodiments, the FTI is administered for at least 6 cycles. In someembodiments, the FTI is administered for up to 12 cycles. In someembodiments, the FTI is administered orally at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle for at least threecycles. In some embodiments, tipifarnib is administered for at least 3cycles. In some embodiments, tipifarnib is administered for at least 6cycles. In some embodiments, tipifarnib is administered for up to 12cycles. In some embodiments, tipifarnib is administered orally at a doseof 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatment cyclefor at least three cycles.

In some embodiments, provided herein is a method of treating a HNSCC ina subject with tipifarnib based on the presence of a H-Ras mutation. Insome embodiments, the HNSCC can be HPV negative HNSCC. In someembodiments, the HNSCC can be relapsed/recurrent HNSCC. In someembodiments, the HNSCC can be metastatic HNSCC. The method providedherein includes (a) determining the presence or absence of a H-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of a tipifarnib to thesubject if the sample is determined to have a H-Ras mutation. The samplecan be a tumor sample. In some embodiments, the methods include (a)determining a HNSCC patient to have a H-Ras mutation, and subsequently(b) administering a therapeutically effective amount of a tipifarnib tothe subject.

In some embodiments, provided herein is a method of treating a salivarygland cancer in a subject with tipifarnib based on the presence of aH-Ras mutation. In some embodiments, the salivary gland cancer can beadvanced salivary gland cancer. In some embodiments, the salivary glandcancer can be metastatic salivary gland cancer. The method providedherein includes (a) determining the presence or absence of a H-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of tipifarnib to thesubject if the sample is determined to have a H-Ras mutation. The samplecan be a tumor sample. In some embodiments, the methods include (a)determining a salivary gland cancer patient to have a H-Ras mutation,and subsequently (b) administering a therapeutically effective amount oftipifarnib to the subject. In some embodiments, the methods includeadministering the subject with another FTI described herein or otherwiseknown in the art. In some embodiments, the FTI is selected from thegroup consisting of tipifarnib, arglabin, perrilyl alcohol, lonafarnib(SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176,AZD3409, and BMS-214662.

In some embodiments, provided herein is a method of treating a thyroidcancer in a subject with tipifarnib based on the presence of a H-Rasmutation. In some embodiments, the thyroid cancer can berelapsed/recurrent thyroid cancer. In some embodiments, the thyroidcancer can be metastatic thyroid cancer. In some embodiments, thethyroid cancer can be advanced thyroid cancer. The method providedherein includes (a) determining the presence or absence of a H-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of tipifarnib to thesubject if the sample is determined to have a H-Ras mutation. The samplecan be a tumor sample. In some embodiments, the methods include (a)determining a HNSCC patient to have a H-Ras mutation, and subsequently(b) administering a therapeutically effective amount of tipifarnib tothe subject. In some embodiments, the methods include administering thesubject with another FTI described herein or otherwise known in the art.In some embodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the methods include (a) determining a HNSCC patientto have a H-Ras mutation, and subsequently (b) administering tipifarnibto the subject, wherein the tipifarnib is administered orally at a doseof 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatment cycle.In some embodiments, the HNSCC patient has relapsed/refractory HNSCC. Insome embodiments, the HNSCC patient has HPV negative HNSCC.

In some embodiments, the methods include (a) determining a salivarygland cancer patient to have a H-Ras mutation, and subsequently (b)administering tipifarnib to the subject, wherein the tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle.

In some embodiments, the methods include (a) determining a thyroidcancer patient to have a H-Ras mutation, and subsequently (b)administering tipifarnib to the subject, wherein the tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle.

In some embodiments, the methods further comprise administering a secondtherapy to the patient having a solid tumor with a H-Ras mutation. Insome embodiments, the second therapy is a chemotherapy, such ascisplatin, 5-FU, carboplatin, paclitaxel, or platinum-based doublet(e.g., cisplatin/5-FU or carboplatin/paclitaxel). In some embodiments,the second therapy is an anti-EGFR antibody therapy (e.g. Cetuximab,Panitumumab, Afatinib). In some embodiments, the second therapy istaxanes, methotrexate, and/or cetuximab. In some embodiments, the secondtherapy is a radiation therapy. In some embodiments, the second therapyinclude those targeting PI3K pathway: BKM120 (buparlisib)+cetuximab,BYL719+cetuximab, Temsirolimus+cetuximab, Rigosertib+cetuximab; thosetargeting MET pathway: Tivantinib+cetuximab, Ficlatuzumab+cetuximab;those targeting EGFR/HER3 pathway Afatinib+cetuximab±paclitaxel,Patritumab; those targeting FGFR pathway: BGJ398; those targetingCDK4/6-cell cycle pathway: Palbociclib, LEE011; RTK inhibitor: Anlotiniband chemotherapy: Oral Azacitidine. In some embodiments, the secondtherapy is an immunotherapy, such as anti-PD1 or anti-PDL1 antibodies.

6. EXAMPLES

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention. All of the references cited to herein areincorporated by reference in their entireties.

Example I Identification of Immunological Biomarkers Associated withClinical Benefit of Tipifarnib

Analyses of gene expression profiling in bone marrow samples from twoAML studies as well as the tipifarnib IC50 data in multiple cells linesrevealed that multiple immunologically related genes, especially NKcells related genes, were associated with better prognosis fortipifarnib. While some of these genes appeared to be non-specific totipifarnib treatment, others including KIR2DS2, KIR2DL2, KIR2DS5,KIR2DL5, and GZMM were specifically associated with clinical benefits oftipifarnib but not other non-FTI chemotherapy agents, such as cytarabineand mitoxantrone.

The current study used microarray data generated from global geneexpression assay of bone marrow samples collected in two clinicalstudies investigating the efficacy and safety of FTI tipifarnib. Oneclinical study was conducted in adult patients aged 65 years or olderwith previously untreated AML, and the other conducted in relapsed andrefractory AML. The clinical results for these studies were previouslypublished (Lancet et al., Blood 109:1387-1394 (2007); Harousseau et al.,Blood 9:9 (2007); Raponi et al., Clin. Cancer Res. 13:2254-2260 (2007)),and the gene profiling data were publically available in NCBI's GeneExpression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and areassessable through GEO Series accession numbers GSE8970 and GSE5122.

As described in Raponi et al., Blood 111:2589-96 (2008), BM samples werecollected from consenting patients before treatment with tipifarnib, andmononuclear cells were processed on site. Total RNA was extracted fromcell samples, quality controlled, and further processed for microarrayanalysis. DNA was isolated from the same sample. Samples were assayedfor global gene expression, and/or quantitative polymerase chainreaction (QPCR) of specific genes).

Response to tipifarnib is reported in the clinical study report and wasdefined as patients who had a complete remission (CR), a partialremission (PR), or hematologic improvement (HI). PR and HI patients wereincluded as responders, since it was previously shown that they had asimilar survival benefit to those achieving a CR. Briefly, CR wasdefined as BM showing less than 5% myeloblasts with normal maturation ofall cell lines, absolute neutrophil count (ANC) of at least 10⁹/L(1000/μL), and a platelet count of 100×10⁹/L (100 000/μL). PR wasdefined as the presence of recovery of ANC and platelets to the abovestated levels, but with 5% to 19% BM blasts, and a greater than 50%decrease in BM blast percentage from baseline. HI was defined as thesame as PR, except with recovery of ANC to 0.5 to 1×10⁹/L (500 to1000/μL) and platelet count to 20 to 100×10⁹/L (20 000 to 100 000/μL).Progressive disease (PD) was defined as any of the following: more than50% increase in BM blast percentage from baseline (more than 5% blastsif baseline is less than 5%, more than 10% blasts if baseline is 5% to10%, and more than 20% blasts if baseline 10% to 20%); greater than 50%increase in circulating blasts; new appearance of circulating blasts onat least 2 consecutive occasions; or development of extramedullaryleukemia. Stable disease (SD) was defined as any response not meetingCR, PR, HI, or PD criteria.

Kaplan Meier curves were used to investigate the relationship betweenbiomarker values and clinical benefit. The identification of multiple NKcell related genes, the long duration of response observed in sometipifarnib patients, and the role of RAS mediated signal transduction inNK cells support that the responsiveness of AML patients to tipifarnibtreatment could be resulted from bone marrow infiltrates of immunecells.

1. Correlation of KIR2DS5 Expression Level with Clinical Benefit ofTipifarnib

As shown in FIG. 1A, the expression level of KIR2DS5 is associated withthe outcome of AML patients treated with tipifarnib. When categorizingpatients based on different clinical responses, it was found that the PDpatients were clustered in the lower end of the KIR2DS5 expressioncontinuum; the CR patients were clustered in the higher end of theKIR2DS5 expression continuum; and the HI and SD patients were clusteredbetween the CR and PD groups.

Additionally, a strong correlation was identified between the expressionlevel of KIR2DS5 and the PFS of AML patients treated with tipifarnib(FIG. 1B). The patients whose KIR2DS5 expression levels were at thehighest (4^(th)) quartile had statistically significant longer PFScompared to the rest of the patients. The correlation supports that AMLpatients can be selected based on the expression level of KIR2DS5 fortipifarnib treatment in order to increase the likelihood ofresponsiveness to the treatment.

2. Specific Correlation Between the Ratio of Expression of KIR2DS2 toKIR2DL2 with Clinical Benefit of Tipifarnib

As shown in FIGS. 2A and 2B, the ratio of expression level of KIR2DS2 tothe expression level of KIR2DL2 (the 2DS2/2DL2 ratio) was stronglycorrelated with both the PFS (FIG. 2A) and the OS (FIG. 2B) of AMLpatients treated with tipifarnib. As shown, the patients with the2DS2/2DL2 ratio at the highest (4th) quartile had statisticallysignificantly longer PFS (median=431 days) and OS (median=750 days)compared to the rest of the patient population.

In addition, the correlation between 2DS2/2DL2 ratio and clinic benefitwas specific to tipifarnib and was not observed for other non-FTIchemotherapy agents, such as cytarabine and mitoxantrone (Chemotherapydata from Metzeler K H et al., Blood, 112:4193-201 (2008)). As shown inFIGS. 3A and 3B, as well as the table below, the survival probability ofAML patients treated with high dose cytarabine and mitoxantrone were notdistinguishable between those with the 2DS2/2DL2 ratio at the highestquintile and the rest of the patients.

5^(th) Q/1-4^(th) Q/all Median Set- Study (days) ting Treatment HRN/5^(th) Q GSE12417- 233/321/294 Front high-dose cytarabine 1.39 163/33GPL96 Line plus mitoxantrone GSE12417- 308/624/538 Front high-dosecytarabine 1.38  79/16 GPL570 Line plus mitoxantrone/ intense chemo in17 pts GSE8970 754/179/233 Front Tipifarnib 0.21 34/7 (CTEP-20) LineEld- erly

The specific correlation between 2DS2/2DL2 ratio and clinic benefit oftipifarnib supports that AML patients can be selected based on the2DS2/2DL2 ratio for tipifarnib treatment in order to increase theoverall response to the treatment.

3. Specific Correlation Between the Ratio of Expression of KIR2DS5 toKIR2DL5 with Clinical Benefit of Tipifarnib

As shown in FIG. 4A, the ratio of expression level of KIR2DS5 to theexpression level of KIR2DL5A (the 2DS5/2DL5 ratio) was stronglycorrelated with both the PFS and the OS of AML patients treated withtipifarnib. As shown, the patients with the 2DS5/2DL5A ratio at thehighest (4th) quartile had statistically significantly longer PFS and OScompared to the rest of the patient population.

In addition, the correlation between 2DS5/2DL5 ratio and clinic benefitwas specific for tipifarnib and not observed for other non-FTIchemotherapy agents, such as cytarabine and mitoxantrone (Chemotherapydata from Metzeler K H et al., Blood, 112:4193-201 (2008)) (FIG. 4B). Asshown in FIG. 4B, the survival probability of AML patients treated withhigh dose cytarabine and mitoxantrone were not distinguishable amongpatients with different 2DS5/2DL5 ratio.

The specific correlation between 2DS5/2DL5 ratio and clinic benefit oftipifarnib supports that AML patients can be selected based on the2DS5/2DL5 ratio for tipifarnib treatment in order to increase theoverall response to the treatment.

4. Specific Correlation of GZMM Expression Level with Clinical Benefitof Tipifarnib

As shown in FIG. 5, the expression level of GZMM is associated with theoutcome of AML patients treated with tipifarnib. When categorizingpatients based on different clinical responses, it was found that the PDpatients were clustered in the lower end of the GZMM expressioncontinuum and had the lowest median expression level of GZMM among thefour groups (CR, HI, SD and PD). The CR patients were clustered in thehigher end of the GZMM expression continuum and had the highest medianexpression level of GZMM among the four groups.

Additionally, a strong correlation was also identified between theexpression level of GZMM and the OS and PFS of AML patients treated withtipifarnib (FIG. 6A). The patients whose GZMM expression levels were atthe highest (4^(th)) quartile had statistically significant longer OSand PFS compared to the rest of the patients. The correlation betweenGZMM expression level and clinic benefit was specific for tipifarnib andnot observed for other non-FTI chemotherapy agents, such as cytarabineand mitoxantrone (FIG. 6B). The specific correlation supports that AMLpatients can be selected based on the expression level of GZMM fortipifarnib treatment in order to increase the overall response to thetreatment.

5. Specific Correlation of KIR2DS2 Expression Level with ClinicalBenefit of Tipifarnib

As shown in FIG. 7A, the expression level of KIR2DS2 is associated withthe outcome of AML patients treated with tipifarnib. A strongcorrelation was identified between the expression level of KIR2DS2 andthe OS of AML patients treated with tipifarnib (FIG. 7A). The patientswhose KIR2DS2 expression levels were at the highest (4^(th)) quartilehad statistically significant longer OS compared to the rest of thepatients (FIG. 7A and FIG. 7B, upper left panel).

As shown in FIG. 7B and the table below, expression of KIR2DS2 stronglycorrelated with clinical benefit, including complete response rate andsurvival endpoints. Patients in the upper (4^(th)) quartile of KIR2DS2expression had a median survival of 564 days whereas those in the1^(st)-3^(rd) quartile of KIR2DS2 expression had a median survival of153 days. In contrast, no correlation was identified between theexpression of NK cell markers, including KIR2DS2, and the clinicalbenefit derived from chemotherapy treatment in a subset of 51 previouslyuntreated and elderly (>65 years) AML patients enrolled in the GermanAML Cooperative Group 1999 study (AMLCG 1999) (FIG. 7B, right panel). Ofthe 34 previously untreated poor-risk and elderly AML patients who weretreated with tipifarnib in a prior Phase 2 clinical trial, 25 had priorMDS. This specific correlation supports that cancer patients can beselected based on the expression level of KIR2DS2 for tipifarnibtreatment in order to increase the likelihood of responsiveness to thetreatment for AML and MDS.

Median KIR2DS2 Low KIR2DS2 High Overall 1^(st)-3^(rd) Quartile 4^(th)Quartile Survival Median Survival (Upper) Median Hazards Treatment (n)(days) (days) Survival (days) Ratio Tipifarnib (34) 233 153 564 0.303Chemotherapy 240 176 284 0.83 (51)

Example II A. Stratification of AML Patients for Tipifarnib ClinicalTrials

A clinical trial can be conducted that includes KIR typing as part ofthe patient inclusion criterion. For example, a study can be conductedfor tipifarnib treatment in AML, patients who are older than 60 orotherwise unfit for standard chemotherapy, or have refractory orrelapsed AML.

Before an AML patient is admitted to the clinical trial, a bone marrowsample or blood sample is obtained from the patient. The sample is thensubjected to microarray analysis. DNA is isolated from the sample ofTrizol-processed bone marrow as per the manufacturer's instructions(Qiagen). Samples are assayed for global gene expression, andquantitative polymerase chain reaction (QPCR) of specific genes,including KIR2DS2, KIR2DL2, KIR2DS5 and KIR2DL5. Among other things, themicroarray analysis provides the genotype of KIR genes of the patient.If the patient is identified to be a carrier of KIR2DS2 gene, or acarrier of KIR2DS5 gene, the patient is then admitted for the tipifarnibtrial. An exemplary inclusion criterion can be as follows:

Pathologic confirmation of the diagnosis of AML (>=20% marrow blasts)

ECOG performance status 0 or 1

Patients must be able to give informed consent

SGOT and SGPT =<2.5× normal limits (grade 1)

Serum creatinine =<1.5× normal limits (grade 1)

AML (any of the following):

-   -   Newly diagnosed AML in adults >=70 years    -   Newly diagnosed AML arising from MDS in adults >=60 years    -   Biopsy-proven relapsed or refractory AML    -   Hyperleukocytosis with >=30,000 leukemic blasts/uL

Carrier of KIR2DS2, or KIR2DS5, confirmed by bone marrow biopsy.

An exemplary dosage regime can be: Patients receive 600 mg tipifarniborally (PO) twice daily (B.I.D.) on days 1-21. Courses repeat every 28days in the absence of disease progression or unacceptable toxicity.

Complete remission (CR) rate and Partial remission (PR) rate can beprimary outcome measures of the trial.

B. MDS Patients for Tipifarnib Clinical Trials with Immune Cell Markersas Secondary Endpoints

A clinical trial can be conducted that includes KIR typing as part ofthe patient inclusion criterion. For example, a study can be conductedfor tipifarnib treatment in AML patients who have MDS, or specificallylower risk MDS. The primary endpoint of the study is transfusionindependence according to the adult Myelodysplastic/MyeloproliferativeNeoplasms International Working Group criteria or related responseassessment system. Secondary endpoints could include the analysis ofimmune cell markers, especially NK cell markers such as KIR2DS2,KIR2DS5, KIR2DL2, KIR2DL5 and GZMM.

When a patient with lower risk MDS is admitted to the clinical trial, abone marrow sample or blood sample is obtained from the patient. Thesample is then subjected to microarray analysis. DNA is isolated fromthe sample of Trizol-processed bone marrow as per the manufacturer'sinstructions (Qiagen). Samples are assayed for global gene expression,and quantitative polymerase chain reaction (QPCR) of specific genes,such as KIR2DS2, KIR2DS5, KIR2DL2, KIR2DL5 and GZMM. Among other things,the microarray analysis provides the genotype of KIR genes of thepatient.

An exemplary dosage regime can be: Patients receive 600 mg tipifarniborally (PO) twice daily (B.I.D.) on days 1-21. Courses repeat every 28days in the absence of disease progression or unacceptable toxicity.

A companion diagnostic test can also be used to aid in the selection ofpatients in clinical trials of tipifarnib in patient population withlower risk MDS. Genetic assays detecting the presence or absence of theKIR genes in NK cells as described herein or otherwise known in the artcan be used. Assays described herein (e.g. a PCR based assay), orotherwise known in the art to determine biomarker expression levels canalso be used, and optimal biomarker cut-off criterion for patientselection can be determined for subsequence clinical studies.

Example III Individualized Treatment Decisions for CMML Patients

The following procedures can be taken to determine whether a CMMLpatient is suitable for an FTI treatment, such as a tipifarnibtreatment.

BM sample is collected from the patient before treatment, andmononuclear cells were processed on site. Total RNA is extracted fromcell samples using the Trizol Kit (Qiagen, Santa Clarita, Calif.). RNAquality is determined by assessing the presence of ribosomal bands on anAgilent Bioanalyzer (Agilent, Palo Alto, Calif.). Good-quality samplesare further processed for microarray analysis.

For each sample, 1 g total RNA (as assessed by OD260) is reversetranscribed using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, Foster City, Calif.) according to themanufacturer's instructions. Samples can then incubated at 25° C. for 10minutes and then 37° C. for 30 minutes for optimum RNA conversion. QPCRis performed using the ABI Prism 7900HT sequence detection system(Applied Biosystems) with all samples run in triplicate. Each reactioncontains 5 μL Taqman Universal PCR Master Mix containinguracil-N-glycosylase (Applied Biosystems), 4.5 μL cDNA template, and 0.5μL of 20× Assay on Demand Gene Expression Assay Mix (Applied Biosystems)or 9 pmol both forward and reverse primer and 2.5 pmol probe in a totalreaction volume of 10 μL. All primer and fluorescein amidite (FAM)fluorogenic probe sets are chosen to generate amplicons less than 100nucleotides, allowing for amplification of transcripts from degraded RNAsamples. Primers and probes are designed for specific amplification ofKIR2DS2 and KIR2DL2. All primer sets span exon boundaries and thusspecifically amplify mRNA transcripts and not genomic DNA.

The KIR2DS2/KIR2DL2 expression ratio is then calculated using methodsknown in the art (e.g. Ma et al., Cancer Cell, 5:607-616 (2004), whichis hereby incorporated by reference in its entirety). The raw Ct valuesare normalized by subtracting the mean Ct from the sample set, dividingby the standard deviation, and then calculating the difference of thenormalized Ct values of each gene.

As described herein, a reference expression ratio of KIR2DS2/KIR2DL2 canbe determined by statistic analysis. As shown in FIGS. 2A and 2B(Example I. II above), for example, a reference expression ratio can bethe expression ratio that distinguish the patients with 2DS2/2DL2 ratioat the highest (4th) quartile from the rest of the patients.Accordingly, if the 2DS2/2DL2 ratio of the CMML patient is determined tobe higher than the reference ratio (namely, the 2DS2/2DL2 ratio of theCMML patient is in the highest (4th) quartile), and the patient is nototherwise prevented from receiving a tipifarnib treatment, then atipifarnib treatment is prescribed. On the other hand, if the 2DS2/2DL2ratio of the CMML patient is determined to be lower than the referenceratio, a tipifarnib treatment is not recommended.

If a tipifarnib treatment is prescribed to the CMML patient, the CMMLpatient can simultaneously receive another treatment, such as ionizingradiation, or a second active agent or a support care therapy, as deemedfit by the oncologist. The second active agent can be aDNA-hypomethylating agent, such as azacitidine or decitabine.

Example IV Lower IC50 for Tipifarnib in Myeloid and Lymphoid Cell Lineswith Wild Type K-RAS/N-RAS Status

As shown in the table below, analyses of tipifarnib IC50 data inmultiple myeloid and lymphoid cells lines revealed that cell linescarrying codon 12, 13 and/or 61 N-RAS or K-RAS mutations are resistantto tipifarnib, while cell lines that do not carry these mutations,including those that have wild type N-RAS and K-RAS are more sensitiveto tipifarnib treatments.

Tipifarnib IC50 for Myeloid and Lymphoid cell lines.

Tipifarnib IC50 Cell Line NRAS KRAS (log μM) ML-2 wt A146T −4.29 SIG-M5wt wt −4.23 QIMR-WIL wt wt −4.12 CMK wt wt −1.89 GDM-1 wt wt −1.04 HELwt wt −0.93 NKM-1 wt wt −0.26 CESS wt wt 0.70 OCI-AML2 wt wt 0.71 KG-1wt wt 1.12 MONO-MAC-6 wt wt 1.21 CTV-1 wt wt 1.52 HL-60 Q61L wt 1.94KMOE-2 Q61R wt 2.14 K052 G13R wt 2.77 NOMO-1 wt G13D 6.84 THP-1 G12D wt7.67 P31-FUJ G12C wt 7.76Data from Genomics of Drug Sensitivity in Cancer (“GDSC”).

Example V Durable Responses in MDS/CMML Patients with N-RAS/K-RAS WildType Tumor Status Treated with Tipifarnib

Twenty-one patients with MDS were treated with tipifarnib in the Phase 1dose escalation study. Tipifarnib was administered twice daily(3-weeks-on/1-week-off schedule for 8 weeks) (starting dosage, 300 mg bymouth twice daily; total, 600 mg).

Objective response 3 HI, 2 PR and 1 CR were observed in 6 of 20 (30%)evaluable patients. As shown in Table 2 below, MDS patients with wildtype N-RAS and K-RAS are more likely to have durable responses (Kurzrocket al., Blood, 102(13):4527-34 (2003)).

Duration Total daily dose, Diagnosis Response (Month) mg Ras MutationRAEB HI 16   300* No CMML HI 2 600 Yes (K-RAS) CMML PR 6 600 Yes (N-RAS)CMML PR 16+ 800 No RAEB HI 3 900 No RAEB-T CR  9+ 800 No *Patientstarted at a total daily dose of 600 mg/d, but dose was reduced after 2weeks. RAEB: refractory anemia with excess blasts.

Example VI Prolonged PFS and Higher Response Rate in AML Patients withN-RAS Wild Type Status Treated with Tipifarnib

CTEP-20 was a phase 2 clinical trial of tipifarnib in previouslyuntreated elderly or unfit AML patients. (Raponi et al., Blood111:2589-96 (2008)).

N-RAS gene status was determined for 32 patients. As shown in FIG. 8, atrend for better PFS was observed in AML patients with wild type N-RAS(PFS=157 days) as compared to those with mutant N-RAS (PFS=65 days). Asshown in FIG. 9, patients with wild type N-RAS (43% ORR) has a higherresponse rate compared to those with mutant N-RAS (27% ORR).Accordingly, AML patients can be selected based on mutation status ofRAS gene for tipifarnib treatment in order to increase theresponsiveness to the treatment.

Example VII Tipifarnib Clinical Trial in CMML Patients Stratified Basedon RAS Mutation Status

This example describes a Phase 2 clinical study of tipifarnib with theprimary objective being to assess the antitumor activity of tipifarnib,in terms of Objective Response Rate (ORR) using theMyelodysplastic/Myeloproliferative International Working Group (MDS/MPNIWG) criteria, of tipifarnib in subjects with chronic myelomonocyticleukemia (CMML) and in subjects with CMML whose disease is KRAS/NRASwild type. Secondary objectives include accessing the effect oftipifarnib on CR rate, complete cytogenetic remission, partialremission, marrow response, and clinical benefit; duration of response;rate of PFS at 1 year; rate of survival at 1 year; adverse event (AE)profile according to National Cancer Institute Common TerminologyCriteria for Adverse Events version 4.03 (NCI CTCAE v 4.03).

This Phase 2 study investigates the antitumor activity in terms of ORRof tipifarnib in subjects with CMML. Up to 20 eligible subjects areenrolled and retrospectively stratified into one of two strata(approximately 10 subjects per stratum) based on subject KRAS and/orNRAS mutational status. The first stratum can enroll subjects with tumorKRAS and NRAS wild type status. The second stratum can enroll subjectswith either tumor KRAS mutant, NRAS mutant or double mutant status.

Subjects can receive tipifarnib administered at a starting dose of 900mg, orally with food, twice a day (b.i.d.) for 7 days in alternatingweeks (Days 1-7 and 15-21) in 28 day cycles. At the discretion of theinvestigator, the dose of tipifarnib can be increased to 1200 mg b.i.d.if the subject has not experienced dose limiting toxicities at the 900mg dose level. Subjects who develop serious adverse events (SAE) or≧grade 2 treatment-emergent adverse events (TEAE) that are deemedrelated to tipifarnib and lasting ≧14 days will not undergo doseescalation. Stepwise 300 mg dose reductions to controltreatment-related, treatment-emergent toxicities are also allowed.

In the absence of unmanageable toxicities, subjects can continue toreceive tipifarnib treatment until disease progression. If a completeresponse is observed, therapy with tipifarnib can be maintained for atleast 6 months beyond the start of response.

Disease assessments (bone marrow, hematology and quality of lifeevaluations) can be performed at screening and at the Day 22 visit (±5days) performed during Cycles 2, 4, 6 and every approximately 12 weeksthereafter (Cycles 9, 12, 15, etc.). Hematologic assessments, includingperipheral blood evaluations and review of transfusion requirements, canbe performed at screening and at least monthly until diseaseprogression. A screening bone marrow aspirate/biopsy is not necessary toinitiate treatment in subjects who have had a bone marrowaspirate/biopsy confirming their diagnosis within 4 weeks prior to Cycle1 Day 1 and can provide samples for the completion of study objectives.If the bone marrow aspirate is inadequate for the scheduled diseaseassessment, a bone marrow biopsy can be performed. Additional disease orhematologic assessments can be conducted if deemed necessary by theInvestigator. The timing of the disease and hematologic assessments aremaintained as much as possible independently of potential treatmentcycle delays.

Example VIII Individualized Treatment Decisions for CMML Patients

The following procedures can be taken to determine whether a CMMLpatient is suitable for an FTI treatment, such as a tipifarnibtreatment.

DNA can be extracted predominantly from bone marrow cells (mononuclearcells or buffy coat) or the peripheral blood of the patient at CMMLpresentation. The mutation status of N-Ras and K-Ras is determined byDNA sequencing, using a fluorescent primer-adapted chainterminationmethod on an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.). When direct sequencing is negative, PCR products are cloned(Original TA Cloning Kit; Invitrogen, Groningen, the Netherlands) andsequenced.

If the CMML patient is determined to have not any mutations at codons12, 13, and 61 of K-Ras or N-Ras, or if the CMML patient is determinedto have wildtype K-Ras and N-Ras, and if the patient is not otherwiseprevented from receiving a tipifarnib treatment, a tipifarnib treatmentis prescribed. On the other hand, if the CMML patient is determined tohave either a N-Ras or K-Ras mutation that results in the activation ofeither N-Ras or K-Ras, a tipifarnib treatment is not recommended.

If a tipifarnib treatment is prescribed to the CMML patient, the CMMLpatient can simultaneously receive another treatment, such as ionizingradiation, or a second active agent or a support care therapy, as deemedfit by the oncologist. The second active agent can be aDNA-hypomethylating agent, such as azacitidine or decitabine.

Example IX Tipifarnib Clinical Trial in Solid Tumor Patients StratifiedBased on HRAS Mutation

A Phase 2 clinical trial was initiated to use tipifarnib in thetreatment of advanced tumors with a known HRAS mutation. The clinicaltrial design includes enrolling 2 cohorts of 18 patients each. Cohort 1enrolls subjects with malignant thyroid tumors with HRAS mutations,independent of thyroid histology. Cohort 2 enrolls any subject with anon-hematological HRAS mutant tumor other than thyroid cancer who meetseligibility criteria.

This clinical trial was designed to include two stages, with the firststage including 11 evaluable patients, and the second stage including 7additional evaluable patients, and a cohort would not proceed to thesecond stage if one or no objective response is observed in a cohort inthe first stage. The clinical trial is considered positive if at least 4responses are observed in a cohort out of 18 subjects. The primaryendpoint is objective response rate, and tumor response assessments areconducted according to the Response Evaluation Criteria in Solid Tumorsversion 1.1 criteria (confirmation of response is required).

According to the protocol, tipifarnib is administered to enrolledpatients at a starting dose of 900 mg, orally with food, twice a day(b.i.d.) for 7 days in alternating weeks (Days 1-7 and 15-21) in 28 daycycles. At the discretion of the investigator, the dose of tipifarnibcan be increased to 1200 mg b.i.d. if the subject has not experienceddose limiting toxicities at the 900 mg dose level. Subjects who developserious adverse events (SAE) or ≧grade 2 treatment-emergent adverseevents (TEAE) that are deemed related to tipifarnib and lasting ≧14 dayswill not undergo dose escalation. Stepwise 300 mg dose reductions tocontrol treatment-related, treatment-emergent toxicities are alsoallowed.

Four (4) evaluable patients were enrolled in the first cohort (patientswith malignant thyroid tumors with HRAS mutations) and eleven (11)evaluable patients were enrolled in the second cohort (patients withnon-hematologic malignancies other than thyroid cancer with HRASmutations). In the second cohort, three (3) of those patients haverelapsed/refractory head-and-neck carcinoma and two (2) of those three(3) experienced confirmed objective partial responses (PR) and a thirdpatient experienced disease stabilization beyond six months (>8 months).All three head and neck carcinoma patients are HPV negative. All headand neck patients remain on study. The responses were observed in two PRpatients after 3 cycles of treatment, six cycles for one and three forthe other. In addition, five (5) patients enrolled patients havesalivary gland cancers with HRAS mutations, and three (3) of themexperienced disease stabilization beyond six months (>7, 9 and >11months). Cohort 2 was proceeded into the second stage of the trial forenrollment of additional seven patients per the trial protocol.

Example X Individualized Treatment Decisions for Solid Tumor Patients

The following procedures can be taken to determine whether a patienthaving a solid tumor is suitable for an FTI treatment, such as atipifarnib treatment. The patient can have a thyroid tumor. The patientcan have a salivary tumor. The patient can also have a head and necktumor. The head and neck tumor can be a head and neck tumor squamouscarcinoma.

DNA can be extracted from tumor sample of the patient. The mutationstatus of H-Ras is determined by DNA sequencing, using a fluorescentprimer-adapted chaintermination method on an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.). When direct sequencing is negative,PCR products are cloned (Original TA Cloning Kit; Invitrogen, Groningen,the Netherlands) and sequenced.

If the patient having a solid tumor is determined to have a mutation atcodons 12, 13, and 61 of H-Ras, or another mutation that results inactivation in H-Ras, and if the patient is not otherwise prevented fromreceiving a tipifarnib treatment, a tipifarnib treatment is prescribed.On the other hand, if the patient is determined to not have any mutationthat results in the activation of H-Ras, or to have wild type H-Ras, atipifarnib treatment is not recommended.

If a tipifarnib treatment is prescribed to the patient, the patient cansimultaneously receive another treatment, such as ionizing radiation, ora second active agent or a support care therapy, as deemed fit by theoncologist. In a head and neck squamous carcinoma patient, theadditional treatment can be an anti-EGFR antibody treatment, ananti-PD1/PDL1 treatment.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 13, 2017, isnamed 14168-020-999_SL.txt and is 42, 924 bytes in size.

We claim:
 1. A method of treating a H-Ras mutant HNSCC in a subject,consisting of administering a therapeutically effective amount oftipifarnib to said subject, wherein said HNSCC is at an advanced stage,metastatic, relapsed or refractory.
 2. The method of claim 1, whereinthe H-Ras mutation of said subject comprises an amino acid substitutionat a codon selected from the group consisting of G12, G13, and Q61. 3.The method of claim 1, comprising determining the presence of H-Rasmutation in a sample from said subject.
 4. The method of claim 3,wherein said sample is a tumor biopsy.
 5. The method of claim 3, whereinsaid H-Ras mutation is determined by a method selected from the groupconsisting of sequencing, Polymerase Chain Reaction (PCR), DNAmicroarray, Mass Spectrometry (MS), Single Nucleotide Polymorphism (SNP)assay, denaturing high-performance liquid chromatography (DHPLC), andRestriction Fragment Length Polymorphism (RFLP) assay.
 6. The method ofclaim 1, wherein tipifarnib is administered at a dose of 1-1000 mg/kgbody weight.
 7. The method of claim 1, wherein tipifarnib isadministered twice a day.
 8. The method of claim 1, wherein tipifarnibis administered at a dose of 600 mg twice a day.
 9. The method of claim1, wherein tipifarnib is administered at a dose of 900 mg twice a day.10. The method of claim 1, wherein tipifarnib is administered for aperiod of one to seven days.
 11. The method of claim 1, whereintipifarnib is administered on days 1-7 and 15-21 of a 28-day treatmentcycle.
 12. The method of claim 1, wherein tipifarnib is administered ata dose of 600 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle.
 13. The method of claim 1, wherein tipifarnib is administered ata dose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle.
 14. The method of claim 1, wherein tipifarnib is administered ata dose of 300 mg twice a day for 3 of 4 weeks in repeated 4 week cycles.15. A method of treating a H-Ras mutant relapsed or refractory head andneck squamous cell carcinoma (HNSCC) in a subject, comprisingadministering a therapeutically effective amount of tipifarnib to saidsubject.
 16. The method of claim 15, wherein the H-Ras mutation of saidsubject comprises an amino acid substitution at a codon selected fromthe group consisting of G12, G13, and Q61.
 17. The method of claim 15,comprising determining the presence of H-Ras mutation in a sample fromsaid subject.
 18. The method of claim 17, wherein said sample is a tumorbiopsy.
 19. The method of claim 17, wherein said H-Ras mutation isdetermined by a method selected from the group consisting of sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) assay, denaturing high-performanceliquid chromatography (DHPLC), and Restriction Fragment LengthPolymorphism (RFLP) assay.
 20. The method of claim 15, whereintipifarnib is administered at a dose of 1-1000 mg/kg body weight. 21.The method of claim 15, wherein tipifarnib is administered twice a day.22. The method of claim 15, wherein tipifarnib is administered at a doseof 600 mg twice a day.
 23. The method of claim 15, wherein tipifarnib isadministered at a dose of 900 mg twice a day.
 24. The method of claim15, wherein tipifarnib is administered for a period of one to sevendays.
 25. The method of claim 15, wherein tipifarnib is administered ondays 1-7 and 15-21 of a 28-day treatment cycle.
 26. The method of claim15, wherein tipifarnib is administered at a dose of 600 mg twice a dayon days 1-7 and 15-21 of a 28-day treatment cycle.
 27. The method ofclaim 15, wherein tipifarnib is administered at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle.
 28. The method ofclaim 15, wherein tipifarnib is administered at a dose of 300 mg twice aday for 3 of 4 weeks in repeated 4 week cycles.
 29. The method of claim15, further comprising administering a therapeutically effective amountof a second active agent or a support care therapy.
 30. The method ofclaim 29, wherein said second active agent is (i) selected from thegroup consisting of a DNA-hypomethylating agent, a therapeutic antibodythat specifically binds to a cancer antigen, a hematopoietic growthfactor, a cytokine, an antibiotic, a cox-2 inhibitor, animmunomodulatory agent, an anti-thymocyte globulin, an immunosuppressiveagent, and a corticosteroid or a pharmacological derivative thereof; or(ii) an anti-PD1 antibody or an anti-PDL1 antibody.